A new method for determination of total phenolic content of four Prunella L. species was developed by immobilizing horseradish peroxidase (HRP) on styrene-divinylbenzene-polygluteraldehyde (STY-DVB- PGA) polymer. The method is based on the spectrophotometric measurement of the final quinone-imine colored product, absorbing at 510 nm, by horseradish peroxidase oxidation in presence of hydrogen peroxide. The effects of solvents were investigated for the extraction of phenolic compounds in Prunella L. plants. HRP was covalently attached on the STY-DVB-PGA with 90% efficiency at optimum immobilization conditions. The total phenol contents ranged from 4 to 152 mg GAE per g of dried sample for pure solvents and ranged from 19 to 763 mg GAE per g of dried sample for acidic extracts using the immobilized enzymatic method. STY-DVB-PGA beads are a good potential support for the immobilization of HRP enzyme and can be used for determination of the total phenolic content of plants. In the analysis of four Prunella L. species, high correlations were obtained between enzymatic and Folin methods, supporting the validity of the enzymatic method proposed. The enzymatic (free/immobilized) methods are more specific and rapid than the Folin method. Acidic solvents are more effective than pure solvents for extraction of phenolic compounds in Prunella L. species.