In this study, the methods of fluorescence spectroscopy and fluorescence quenching titration technique were used to identify the interactions between humic acid (HA) and roxarsone (ROX). Effects of HA concentration, pH and temperature on the bonding strength between HA and ROX were investigated. The results showed that the four fluorescence peaks (E(x)/E(m) = 300 nm/480 nm, 370 nm/480 nm, 420 nm/500 nm, 460 nm/520 nm, marked as peak A, B, C, D respectively) of HA could be quenched by ROX. The extent of decreases in fluorescence intensities of different peaks was different and followed the order of C > B > A > D. The common logarithm of association constants (lg K) between peak A and ROX increased slightly with the increase of HA concentration and were much larger than the bimolecular quenching constant of O2. It was confirmed that the carboxyl groups and the carboxide groups of HA were quenched statically by ROX. The lg K values fluctuated between 3.55 L x mol(-1) and 3.98 L x mol(-1) when pH ranged from 5.00 to 9.00, and the maximum value occurred at pH 6.00. It might be resulted from the fact that pH could change the formation of ROX and conformation of phenolic hydroxyl groups and carboxyl groups in HA. The lg K values decreased and fluctuated between 2.65 L x mol(-1) and 3.89 L x mol(-1) with temperature ranging from 25.0 degrees C to 55.0 degrees C, which further confirmed the static quenching interaction between HA and ROX. Transient-fluorescence spectrum analyses and liner model simulations revealed that single static quenching was the main mechanism between ROX and the functional groups of fluorescence peak A, B, D in HA, and combined dynamic and static quenching was the main mechanism between ROX and the functional groups of peak C in HA.