Fluorescence spectroscopy is a fast, accurate, and non-destructive detection method widely used to detect and analyze pesticide residues. This article applies the three-dimensional fluorescence spectroscopy method combined with the parallel factor (PARAFAC) algorithm to achieve qualitative and quantitative analysis of mixed organic pesticides. Firstly, in different proportions, two kinds of mixed solutions are prepared, including spinosad-lambda-cyfluthrin and spinosad-ningnanmycin. The three-dimensional fluorescence spectra are obtained by scanning with an LS55 fluorescence spectrophotometer; the emission wavelength range is 200~600 nm, and the excitation wavelength range is 250~322 and 260~370 nm, respectively. Then, the parallel factor algorithm is applied to model and analyze the preprocessed spectral data. The predicted spectra and score values of each component in the mixture are obtained through trilinear decomposition, and the predicted spectra are visually matched with the actual spectra to identify pesticide categories. Finally, a linear fitting analysis is conducted between the score value and the concentration of pesticide component, and the mean squared error, coefficient of determination, and recovery rate parameters are calculated. The results showed that the predicted spectra of the pesticide components corresponding to the two mixtures are consistent with their true spectra, especially with high overlap at the fluorescence characteristic peaks. The prediction means that the squared error of each component in the spinosad-lambda-cyhalothrin mixture is 1.985 6×10^-8 and 4.480 0×10^-7, respectively. The corresponding prediction mean squared error of the spinosad-ningnanmycin mixture is 2.155 2×10^-7 and 5.572 2×10^-5, respectively. The model’s coefficient of determination exceeds 0.99, and the average recovery is close to 100%. The test results indicate that three-dimensional fluorescence spectroscopy combined with a parallel factors algorithm can analyze pesticide mixtures with high analytical accuracy. The research content of this article provides a certain methodological basis for the qualitative and quantitative analysis of mixed organic pesticides, and this method can also be extended to the detection and analysis of mixed systems of other types of samples.