In order to solve the alignment problem between the detection head of the large-size photoelectric detection device and the reaction hole, a small, in the traditional chemiluminescence enzyme immunoassay method. A multi-mode optical fiber combined with the optical fiber-type photon counting probe is used to detect the target signal, and an aflatoxin B1 optical fiber detection system based on photoelectric detection technology is designed. The ZEMAX simulation and test results show that the optical fiber photon collection ratio reaches more than 95.83% and RSD ≤ 0.21% in the reaction hole range compared with the reaction hole center position detection, and the alignment requirements of the optical fiber detection port and the reaction hole are greatly reduced. The experimental results showed that the detection limit of the fiber optic detection system was 0.63 μg/L. The correlation coefficient of the standard curve was 0.9977. The RSD was 1.03~2.38 %, and the sample spiked recovery was 92.62~96.03 % with higher linearity, precision, and reproducibility.