To develop a low-cost and high-sensitivity device for detection of aflatoxin B1 (AFB1), an optical fiber immunosensor based on the functionalization of polyaniline for the detection of AFB1 was developed. A sensitized optical fiber sensor for the detection of AFB1 was designed by combining the excellent optical properties of polyaniline (PAni) with the soft and bending-resistant properties of the plastic optical fiber (POF). First, PAni was used to carboxylate the POF surface in the sensing region, and second, the sensing region was cross-linked with glutaraldehyde to immobilize the AFB1 antibody molecule, which could then be obtained by the sensitized optical fiber sensor for detecting AFB1. The sensor can detect the concentration of AFB1 through the change in the light intensity signal of the optical fiber output caused by the immune response. The sensitizing effect of the polyaniline coating on the sensor was experimentally investigated, and the specificity, immunity to interference, and detection limit of the sensor were tested. The results showed that there was a linear relationship between the output signal of the sensor and the AFB1 concentration in the range of 0.01~10μg/L, and the detection limit was 0.53μg/L. The recoveries of the spiked standard ranged from 95.97% to 113.13%, and the specificity and immunity of the sensor were good, thus meeting the needs of a refined detection of AFB1.