Liquid-catalyst fuel cell (LCFC) is a novel device that can convert biomass to electricity directly under mild conditions, but its optimal operational parameters and corresponding mechanism have not been disclosed. In this study, the effect of anolyte pH and temperature on LCFC performance was analyzed comprehensively. Polarization curves and power density curves were measured using the linear sweep voltammetry to evaluate the power generation from LCFC. The Mo⁵⁺ concentration and total organic carbon in anolyte were measured to deduce the redox reaction between organics and heteropoly acid, and the Mo⁵⁺ concentration-absorbance standard curve was established using the potassium permanganate titration method and the ultraviolet visible spectrophotometry. The valence composition of Mo in anolyte was characterized using XPS spectra. The results demonstrated that a proper anolyte acidification significantly promoted the redox reaction between organic substrates and heteropoly acid, but an excessive acidification destroyed the structure of heteropoly acid and accordingly reduced its oxidizability. When the pH of anolyte was less than 1.5, the pH affected the power output of LCFC significantly. A moderate decrease of anolyte pH improved the power density greatly. At pH 0.86, the power density reached the maximum 14.85 mW•cm^–2, which was 1.24 times higher than that without anolyte acidification. At extremely low pH, the power density deteriorated due to the decomposition of heteropoly acid and its weakened oxidizability. At the pH range of 1.5 to 7.5, the effect of pH on LCFC performance was relatively small. Moderate operational temperatures could enhance the performance of LCFC, but excessively high temperatures would dehydrate proton exchange membranes and consequently hindered proton transfer. Therefore, the temperature 80 ℃ was recommended for LCFC operation.