In this paper, a simple, fast, and inexpensive method is introduced for the simultaneous spectrophotometric determination of crystal violet (CV) and malachite green (MG) contents in aquatic samples using partial least squares regression (PLS) as a multivariate calibration technique after preconcentration by graphene oxide (GO). The method was based on the sorption and desorption of analytes onto GO and direct determination by ultraviolet-visible spectrophotometric techniques. GO was synthesized according to Hummers method. To characterize the shape and structure of GO, FT-IR, SEM, and XRD were used. The effective factors on the extraction efficiency such as pH, extraction time, and the amount of adsorbent were optimized using central composite design. The optimum values of these factors were 6, 15 min, and 12 mg, respectively. The maximum capacity of GO for the adsorption of CV and MG was 63.17 and 77.02 mg g(-1), respectively. Preconcentration factors and extraction recoveries were obtained and were 19.6, 98% for CVand 20, 100% for MG, respectively. LOD and linear dynamic ranges for CVand MG were 0.009, 0.03-0.3, 0.015, and 0.05-0.5 (mu g mL(-1)), respectively. The intra-day and inter-day relative standard deviations were 1.99 and 0.58 for CV and 1.69 and 3.13 for MG at the concentration level of 50 ng mL(-1), respectively. Finally, the proposed DSPE/PLS method was successfully applied for the simultaneous determination of the trace amount of CV and MG in the real water samples.