Investigations both on the binding properties and mechanisms of parent polycyclic aromatic hydrocarbons (PAHs) and alkyl PAHs with humic acid (HA) are crucial to understand the environment behavior and bioavailability of PAHs in aquatic ecosystems. Thus, a novel approach for in situ investigation of the interactions between parent PAHs and alkyl PAHs, exemplified by Phenanthrene (Phe), 9-Ethylphenanthrene (9-EP) and Retene (Ret), and HA was established by using fluorescence quenching method with laser-induced nanosecond time-resolved fluorescence (LITRF). By changing the delay time of 50 ns, the fluorescent interference of HA can be eliminated effectively and the concentrations of free Phe, 9-EP and Ret can be determined directly. The binding characteristics of the dissolved HA and Phe, 9-EP and Ret were described by Freundlich nonlinear isothermal model. The results of the model parameters and the single point binding coefficients KOC of Phe, 9-EP and Ret with HA by LITRF quenching method were consistent with those of the conventional fluorescence quenching method. Nonlinear bindings of Phe, 9-EP and Ret to HA were also observed because of the parameter n values below 1, and the degree of nonlinearity of Phe was lower than 9-EP and Ret duo to the n value of Phe larger than 9-EP and Ret. At a same given equilibrium concentration, the KOC of Phe was lower than 9-EP and Ret, and that of 9-EP was near to Ret. The binding affinity of the three PAHs increased with decreasing the equilibrium concentration. The binding characteristics of PAHs with HA largely depends on their hydrophobicity, substituent groups and its ability to fit into hydrophobic cavities in HA. The fluorescence lifetimes of Phe, 9-EP and Ret in presence of HA were 36.90, 35.34 and 35.13 ns, meanwhile the fluorescence lifetimes of Phe, 9-EP and Ret in absence of HA were 36.36, 35.34 and 35.84 ns. There was no significant difference of three PAHs fluorescence lifetime with or without HA, indicating the quenching mechanism for Phe, 9-EP and Ret with HA were primarily static quenching. The LITRF quenching method could be used to in situ explore the interactions between PAHs and HA, which could be contribute to realize the PAHs risk assessment in real time.