Horseradish peroxidase represents one of the most exploited enzymes in the process of enzymatic phenol removal from aqueous solutions. It has a catalytic ability over a broad pH range, temperature and contaminant concentration. In this study we have investigated the influence of pH and temperature on process of phenol removal by crude horseradish peroxidase from aqueous solution. Reaction was performed in the presence of low molecular polyethylene glycol (PEG 300) at different temperatures (4, 12, 17, 20, 25, 30, 35, 40 and 45 °C) and pH values (3, 4, 5, 6, 7, 8 and 9). Reaction was monitored by measuring of absorbance changes of the samples taken at certain time intervals from reaction mixture. Obtained results shown that phenol removal from aqueous solution increases by temperature increase up to 35 °C, after which this effect no longer exists. Also, phenol removal increases in the pH range of 3 – 7, while a further increase of pH value leads to the opposite effect. Based on this it can be concluded that phenol removal from aqueous solutions greatly depends of peroxidase activity, because this temperature and pH values represents the optimum values of peroxidase enzymatic activity.