A dual-label time-resolved fluoroimmunoassay (TRFIA) for simultaneous quantification of aflatoxin B1 (AFB1) and ochratoxin A (OTA) is described. For this, microtitration wells were coated with AFB1-horse radish peroxidase (HRP) and OTA-bovine serum albumin. The standards and samples were loaded on the coated plates, and diluted antibodies and Eu3+- and Sm3+-labeled IgG were then added. Our results showed that the sensitivity of TRFIA for AFB1 was 0.02 microg/L (range 0.02-100 microg/L). The intra- and inter-batch coefficient of variation (CV) was 3.2 and 7.3%, respectively, and the average recovery rate was 88.1%. On the other hand, the sensitivity of OTA was 0.05 microg/L (range 0.05-50 microg/L), the intra- and inter-batch CV was 2.9 and 7.9%, respectively, and the average recovery rate was 89.9%. In the AFB1/OTA-TRFIA, AFB1 and OTA did not mutually interfere. The correlation coefficients between the dual-label AFB1/OTA-TRFIA and the single-label AFB1-TRFIA or OTA-TRFIA were 0.972 and 0.981, respectively, indicating that the results were consistent. Our study suggests that AFB1/OTA-TRFIA allows the simultaneous detection of AFB1 and OTA; is a simple, fast, and economic method for screening large quantities of samples, and has good prospects of application.