Humic substances (HS) are one of the most important parts of dissolved organic matter which are ubiquitous in environments playing an important role in many (bio)geochemical processes. In order to define the effects of environmental conditions on the biological transformation of compounds in the presence of HS, microbial reduction rates of ferrihydrite under different HS concentrations, environmental temperature, and pH values were determined. The results showed that increasing the concentration of standard humic substances (Pahokee peat standard humic acids (PPHA) or Pahokee peat standard fulvic acids (PPFA)) purchased from the International Humic Substances Society could compensate for the certain gap of reduction rates between low temperature (10 °C) and high temperature (35 °C). Furthermore, PPHA showed a greater stimulation of Fe(III) reduction than PPFA in common groundwater temperature (10–25 °C). The reduction rates decreased significantly when pH values were greater than 7.7 with total organic carbon (TOC) contents above 5 mg C/L, and protein-like fluorophore was detected here. When pH values were lower than 7.7, the PPHA fluorescence characteristic primarily showed humic-like fluorophore composed of quinone-like fluorophore, which corresponds to a high microbial reduction rate. The finding of this study is that fluorophore characteristics of HS linked to the microbial reduction rate under different environment conditions are able to provide a potential development for in-suit remediation process prediction.