Fuberidazole and pirimicarb are carbamate pesticides for insect control. There is pesticide accumulation through the food chain that may ultimately migrate into the human body through the crayfish. Here a rapid method for simultaneous determination of fuberidazole and pirimicarb in crayfish is reported. The excitation-emission fluorescence matrix was obtained by simple pretreatment of crayfish meat using acetonitrile, and qualitative and quantitative information was separated from overlapping spectra using the self-weighted alternating trilinear decomposition. The detection limits of the pesticides for fuberidazole and pirimicarb were 0.068 mg/kg and 0.160 mg/kg, respectively, and the limits of quantitation were 0.207 mg/kg and 0.485 mg/kg. The average recoveries were 90.79% and 98.77%. The results show that a combination of excitation-emission fluorescence matrix and alternating trilinear decomposition can be used for the determination of fuberidazole and pirimicarb in crayfish with simple pretreatment using acetonitrile.