A novel method was proposed for the simultaneous determination of pefloxacin and ofloxacin in the environmental water using three-dimension fluorescence spectra coupled with second-order calibration method. All the samples were measured at excitation wavelengths from 230 nm to 400 nm and the emission wavelengths from 360 nm to 580 nm. A three-way data array was generated and then subjected to dealing with the alternating trilinear decomposition algorithm, thus resulting in relative excitation, emission and concentration profiles related to target components and unknown interference(s). These results suggested that a univariate calibration curve associated with component of interest was obtained by the relative concentrations vs the actual concentrations in calibration samples, and further used to predict content in test samples. The average recoveries of pefloxacin and ofloxacin were (101.1±5.3)% and (99.7±4.7)%, with limit of detection being 2.14, 4.34 ng/mL, and limit of quantitation 6.49, 13.16 ng/mL, respectively. The proposed method could be applied to simultaneous determination of pefloxacin and ofloxacin in the environmental water even in the presence of unknown interference(s), and realize the second-order advantage.