Simultaneous biosorption properties of three cationic dyes (Methylene blue, Crystal violet and Safranin) on Sargassum glaucescens were studied. In the most of previous papers in the field of dye biosorption, one dye or dyes with nearly separate spectra were used and dye concentration was determined by Beer’s law at different λmax. Significant of this study is application of dyes with highly overlapped spectra (as many real situations) that their concentrations can be determined by chemometric methods. Plackett–Burman design was applied to identify the most significant factors, Box–Behnken design was used to determine optimal conditions, and principal component-wavelet neural network was used for the simultaneous determination of dye concentrations in ternary solutions. The optimum biosorption conditions were determined as dye concentration 10−4 mol L−1, biosorbent dosage 0.1 g L−1 and biosorbent particle size 0.188 μm. At this condition, maximum biosorption capacity was 0.80 mmol g−1. The biosorption process was slightly slower in the ternary system comparing with single system which was related to competition phenomena between dyes. It was found that the overall biosorption data were described by the pseudosecond- order kinetic model. Fourteen isotherm models were applied to experimental data, and