A novel approach is proposed for direct quantitative analysis of thiabendazole in the orange extract by using excitation-emission matrix fluorescence coupled with second-order calibration methods based on the alternating trilinear decomposition (ATLD) and the alternating normalization-weighted error (ANWE) algorithms, respectively. The average recoveries of thiabendazole in the orange extract by using ATLD and ANWE with an estimated component number of two were 99.7 ± 3.3% and 103.5 ± 4.1%, respectively. Furthermore, the accuracy of the two algorithms was also evaluated through elliptical joint confidence region (EJCR) tests as well as figures of merit, such as sensitivity (SEN), selectivity (SEL) and limit of detection (LOD). The experimental results demonstrate that both algorithms have been satisfactorily applied to the determination of thiabendazole in orange extract, and the performance of ANWE is slightly better than that of ATLD. excitation-emission matrix fluorescence, alternating trilinear decomposition, alternating normalization-weighted error, orange extract, thiabendazole