A reagent kit for detecting ochratoxin A and aflatoxin B1 at the same time and method thereof are provided, which belongs to fields of time-resolved fluoroimmunoassay. Micro plate is coated with OTA-BSA, AFB1-BSA, adding OTA, AFB1 standard or sample, and then adding OTA monoclonal antibody, AFB1 polyclonal antibody. Dissociated OTA, AFB1 and OTA-BSA, AFB1-BSA on micro plates competing corresponding antibody, and antibody which haven't be connected is eliminated after washing, adding Sm3+-goat anti rat and Eu3+- goat anti rabbit, labeled antibody haven't be connected after immune reaction are eliminated after washing. After adding enhancement solution, detecting Eu and scythe fluorescence intensity cps respectively which are inversely proportional to intensity of OTA and AFB1 in sample respectively by time resolved luminoscope, comparing standard curve and the content of OAT and AFB1 in sample is determined. The reagent kits of present invention has merits of simple structure, convenient operation and low cost, two examining results of OAT and AFB1are obtained by one operation, the present invention is used for detecting of grain, feed and food.