© 2019 Fumonisin B1 is the most prevalent member of a family of toxins, known as fumonisins, which occurs mainly in maize, wheat and other cereals. Due to its hepatotoxic and nephrotoxic in all animal species, very strict regulations have been imposed on the levels of fumonisin B1 in cereal and cereal-based foods worldwide. In this work, a rapid determination method of fumonisin B1 by membrane solid phase extraction coupled with solid-phase fluorescence analysis is developed. A rhodamine based fluorescent probe was used for derivatization with fumonisin B1. After derivatization and extraction by nylon membrane, the enriched fumonisin B1 can be detected directly on the membrane without further elution process that is placed in a designed spectra collection device. The established method showed a linear relationship in concentration range of 0.5–5.0 μg/L, with the R2 = 0.991, and a limit of detection of 0.119 μg/L. Method accuracy was further confirmed using LC-MS method by comparing the detection results of 3 corn powder samples spiked with FB1, that demonstrated equivalent results. The results of this study indicated that the proposed method was simple, sensitive, reliable and suitable for trace fumonisins B1 quantitation in corn-based feeds.