Abstract: Microchannel resistive biosensing can be expected to detect the concentration of polystyrene (PS) microspheres in solutions, thus quantifying the target analytes. The promising potential application can be introduced to test the agricultural products, due to its low-cost characteristics and high sensitivity. It is the high demand for automated and highly stable equipment. In this study, an automated detection device was developed to integrate the microchannels, a hardware control system, and a human-machine interface (HMI). Its feasibility was also explored in the detection of the umami substances. Firstly, the high-precision microchannels were fabricated for the high accuracy of the channel structure using 3D micro-nano printing. Secondly, the hardware control system was utilized to coordinate the various units during sample analysis, including automatic sampling, measurement display, cleaning and sample changing, and power management. The relative errors were reduced with the manual operations and detection efficiency. Additionally, a filter module was incorporated to minimize the impact of the external factors on the detection, such as electromagnetic interference and flow rate disturbances, particularly for the data reliability. Lastly, an HMI was designed to drive the entire control system, thus enabling data processing, display, and storage. While the real-time feedback was realized to monitor the detection. The coefficient of variation (CV) was employed to evaluate the effects of the microchannel inner diameter, microchannel length, and sample flow rate on the signal stability. A response surface method (RSM) was conducted to optimize these parameters. The optimal combination of the working parameters was identified as an inner diameter of 100 μm, a length of 2 mm, and a flow rate of 2 mL/min. The stability of the detection was enhanced after optimization. Furthermore, the phosphate-buffered saline (PBS) solution was taken as the blank control group in order to evaluate the performance of the developed device. Polystyrene (PS) microsphere solutions were mixed uniformly with the concentrations ranging from 1 pg/mL to 1μg/mL. The current values were recorded for the different concentrations. As the concentration of the microsphere solution increased gradually, a high correlation was observed between the current and the concentration of PS microspheres in the range of 1 to 1×106 pg/mL, with a detection limit of 0.76 pg/mL. Furthermore, the maximum coefficient was just 1.13% over seven consecutive days, where the maximum coefficient was only 1.8% among three microchannels. These results indicated that the device also exhibited high stability and repeatability after detection. Additionally, the monosodium glutamate (MSG) samples were prepared to verify the feasibility of the microchannel resistive biosensor. The developed device was then used to detect the umami substance, according to the competitive immunoassay reactions. The results showed that the current difference also increased after detection, as the MSG concentration increased gradually. There was a positive correlation between the current difference and the MSG concentration. Taking the monosodium glutamate (MSG) as a representative substance, the linear range of 1 to 1×104 pg/mL was determined with a limit of detection (LOD) of 0.24 pg/mL in the practical testing. The better performance was achieved, including the particle counters, green spectrophotometric approaches, and high-performance liquid chromatography (HPLC). The practical utility of the equipment was validated after comparison. The findings can provide technical support to rapidly and accurately detect quality and safety monitoring of agricultural products.