Abstract: Excessive use of oxytetracycline (OTC) has posed significant threats to microbial communities, soil quality, and public health via the food chain. It can often accumulate in soil from protected agricultural facilities. Therefore, there is an urgent need to develop rapid, sensitive, and cost-effective detection for the OTC residues. In this study, a photoelectrochemical (PEC) aptasensor was constructed using biochar/zinc oxide (ZnO@BIO-C) nanocomposites for the ultrasensitive detection of OTC residues in soil. The BIO-C support was derived from Magnolia grandiflora leaves via a microwave-assisted pyrolysis, followed by hydrothermal synthesis to grow ZnO nanoparticles onto the BIO-C surface. Various techniques were used to characterize the nanocomposites, including scanning electron microscopy, transmission electron microscopy, X-ray diffraction, Raman spectroscopy, and X-ray photoelectron spectroscopy. It was found that the ZnO nanoparticles were uniformly dispersed on the layered BIO-C matrix without significant agglomeration. The BIO-C matrix was introduced to significantly enhance the visible light absorption range for 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, indicating superior PEC performance. A PEC aptasensor was then fabricated to immobilize an OTC-specific aptamer onto the ZnO@BIO-C-modified indium tin oxide electrode. The sensing mechanism relied on the specific recognition between the aptamer and OTC, leading to the dissociation of the OTC-aptamer complex from the electrode surface. The steric hindrance was reduced to restore the electron transfer pathway, resulting in a concentration-dependent increase in photocurrent. The PEC aptasensor exhibited a wide linear range from 1.0 × 10⁻¹² to 5.0 × 10⁻⁸ mol/L under optimal conditions, with a low detection limit of 3.3 × 10⁻¹³ mol/L (S/N = 3). There was excellent selectivity against common coexisting substances, such as tetracycline, salicylic acid, and indole-3-acetic acid. The sensor was also attributed to the high specificity of the aptamer recognition element. The practical applicability of the aptasensor was evaluated using the soil samples from a greenhouse. The sensor with the standard addition achieved high recovery rates from 99.92% to 100.10%, compared with the standard deviations below 5.5%. The sensor fully met the performance requirements for trace antibiotic detection in soil, indicating the high accuracy, reliability, and resistance to matrix interference. A green and sustainable strategy was obtained to convert the agricultural waste into high-value functional materials for environmental monitoring. The ZnO@BIO-C nanocomposite, derived from Magnolia grandiflora leaves, provided an excellent photoactive substrate with enhanced charge separation and visible light response. The PEC sensor was integrated with aptamer technology for the trace detection of antibiotic residues. Nevertheless, the aptamer immobilization currently relies on physical adsorption, thus leading to gradual desorption and low long-term stability. The structural properties of biochar, such as pore size distribution and defect density, were highly dependent on pyrolysis parameters for the performance consistency. Moreover, a more systematic investigation is often required for the interfacial charge transfer kinetics and the influence of soil matrix variability on sensor response. Future research should focus on more robust aptamer anchoring strategies, biochar synthesis parameters, the sensor against standard analytical techniques, and its applicability to a wider range of emerging contaminants. Overall, this work can provide a promising technical pathway for on-site monitoring of antibiotic pollution in agricultural environments. The low-cost, biomass-derived materials can also be expected for advanced sensing applications.