Abstract: The excessive use of oxytetracycline (OTC) in protected agricultural practices has led to its accumulation in facility soils, posing significant threats to microbial communities, soil quality, and public health through food chain transfer. Developing rapid, sensitive, and cost-effective detection methods for OTC residues is urgently needed. In this study, a novel photoelectrochemical (PEC) aptasensor was successfully constructed based on a biochar/zinc oxide (ZnO@BIO-C) nanocomposite for the ultrasensitive detection of OTC in facility soil. The BIO-C support was derived from Magnolia grandiflora leaves via a microwave-assisted pyrolysis process, followed by hydrothermal synthesis to grow ZnO nanoparticles onto the BIO-C surface. Various characterization techniques, including scanning electron microscopy, transmission electron microscopy, X-ray diffraction, Raman spectroscopy, and X-ray photoelectron spectroscopy, confirmed the successful formation of the composite, with ZnO nanoparticles uniformly dispersed on the layered BIO-C matrix without significant agglomeration. The introduction of BIO-C significantly enhanced the visible light absorption range and facilitated the separation and transport of photogenerated charge carriers, effectively suppressing electron-hole recombination. Photoelectrochemical measurements revealed that the ZnO@BIO-C composite with an optimal BIO-C doping ratio of 2% exhibited a photocurrent intensity 2.6 times higher than that of pristine ZnO, demonstrating superior PEC performance. Based on this optimized material, a PEC aptasensor was fabricated by immobilizing an OTC-specific aptamer onto the ZnO@BIO-C-modified indium tin oxide electrode. The sensing mechanism relies on the specific recognition between the aptamer and OTC, which leads to the dissociation of the OTC-aptamer complex from the electrode surface. This dissociation reduces steric hindrance and restores the electron transfer pathway, resulting in a concentration-dependent increase in photocurrent. Under optimized conditions, the proposed PEC aptasensor exhibited a wide linear detection range from 1.0 × 10⁻¹² to 5.0 × 10⁻⁸ mol/L, with a low detection limit of 3.3 × 10⁻¹³ mol/L (S/N = 3). The sensor also demonstrated excellent selectivity against common coexisting substances such as tetracycline, salicylic acid, and indole-3-acetic acid, attributed to the high specificity of the aptamer recognition element. To evaluate its practical applicability, the aptasensor was employed to detect OTC in real facility soil samples collected from a greenhouse. Using the standard addition method, the sensor achieved satisfactory recovery rates ranging from 99.92% to 100.10%, with relative standard deviations below 5.5%. These results comply with the performance requirements specified for trace antibiotic detection in soil, confirming the sensor's accuracy, reliability, and resistance to matrix interference. This study successfully demonstrates a green and sustainable strategy for converting agricultural waste into high-value functional materials for environmental monitoring. The ZnO@BIO-C nanocomposite, derived from Magnolia grandiflora leaves, provides an excellent photoactive substrate with enhanced charge separation and visible light response. Integrated with aptamer technology, the constructed PEC sensor offers a powerful platform for trace detection of antibiotic residues. Certain limitations should be acknowledged. The aptamer immobilization currently relies on physical adsorption, which may lead to gradual desorption and affect long-term stability. The structural properties of biochar, such as pore size distribution and defect density, are highly dependent on pyrolysis conditions and require further optimization for better performance consistency. Moreover, the detailed interfacial charge transfer kinetics and the influence of soil matrix variability on sensor response warrant more systematic investigation. Future research should focus on developing more robust aptamer anchoring strategies, optimizing biochar synthesis parameters, validating the sensor against standard analytical methods, and expanding its applicability to a broader range of emerging contaminants. Overall, this work provides a promising technical pathway for on-site monitoring of antibiotic pollution in agricultural environments and offers new insights into the design of low-cost, biomass-derived materials for advanced sensing applications.