The fluorescence quenching method using laser-induced nanosecond time-resolved fluorescence (LITRF) combined with parallel factor (PARAFAC) analysis was developed for in situ investigation of the interactions of two typical polycyclic aromatic hydrocarbons (PAHs), pyrene (Pyr) and phenanthrene (Phe) either singly or in a mixture with Aldrich humic acid (HA). The concentration of free Pyr and Phe cannot be determined directly at the same time because the LITRF spectra of Pyr, Phe and HA are overlapping at the excitation wavelength of 266 nm. The fluorescent interference of HA can be eliminated quickly and effectively, while the fluorescence intensity and fluorescence decay curves of Pyr and Phe can be acquired by LITRF-PARAFAC analysis. The binding characteristics of PAHs with HA were described with Freundlich isothermal model. Nonlinear binding (n<1) and competitive sorption of Pyr and Phe on HA were verified, which showed that the linearity increased (n tend to be 1) and the single point binding coefficient decreased after the addition of Pyr or Phe. The degree of competition was depended on the concentrations of cosolutes, and competition was much stronger in the case of relatively high competitor concentrations. In addition, the results indicated that the binding characteristics of single component Pyr and Phe with HA obtained with LITRF-PARAFAC quenching method were in accordance with conventional fluorescence quenching method. The quenching mechanism for Pyr and Phe by HA was primarily static quenching which was also verified by quenching rate constant and fluorescence lifetime analyses. The LITRF-PARAFAC quenching method could be used for in situ investigate the interactions between mixture of PAHs and HA, which could be applied to predict and evaluate the environmental behavior and ecological risk of PAHs in real time.