A sensitive and rapid spectrofluorimetric method has been proposed for the quantitative analysis of linear alkylbenzene sulfonate (LABS) and optical brightener (OB) in the presence of unknown interferences with intense spectral overlapping in laundry powders and environmental samples. The method is based on second-order multivariate calibration applying three-way chemometric methods. Due to unique solutions of the three-way data analysis methods and analogy between the structure of LABS and OB, it is apparent that these methods can resolve overlapping signals into pure spectra and relative concentration profiles. Satisfactory recoveries for the spiked laundry powders for LABS (92.8–109.3%) and OB (97.1–105.2%) indicate the high accuracies of the proposed calibration methods for the assessment of LABS and OB in laundry powders. In the case of environmental samples, recoveries of 98.0–109.3% for LABS and 94.0–107.7% for OB show that the present method successfully faces this complex challenge without the necessity of applying separation steps.