The first order derivative synchronous fluoremetry was proposed for simultaneous determination of sunset yellow and ponceau 4R. The effect of different experimental conditions, such as different pH for character of fluorescence spectra and the choosing of the optimal wavelength difference were studied. It was showed that the zero-crossing points were at 313.6 nm for ponceau 4R and at 302.8 nm for sunset yellow in first order derivative synchronous fluorescence spectra. Therefore, 313.6 and 302.8 nm were selected for the determination of sunset yellow and ponceau 4R when Δλ=130 nm. This method could minimize interference without preseparation. The linear ranges of sunset yellow and ponceau 4R were from 0.1 to 2.0 mg·L-1 and from 0.1 to 4.0 mg·L-1 with correlation coefficient 0.996 6 and 0.999 2, the detection limits were 0.041 and 0.019 mg·L-1, RSDs were 4.6% and 4.8% (n=6), respectively. The recoveries varied from 91.0% to 110%. The proposed method was successfully applied in simultaneous determination of sunset yellow and Ponceau 4R in food.