Microbial degradation of phenolic pollutants in industrial wastewater is dependent on enzymatic pathway comprising a cascade of phenol metabolizing enzymes. Phenol hydroxylase is the first enzyme of the pathway catalyzing the initial attack on phenol in green algae Chlorella pyrenoidosa. The present work reports cost effective production of partially purified microalgal phenol hydroylase by single step purification and characterization of its kinetic properties with the view of application for enzyme based remediation of phenolic wastewater or in phenolic biosensor. The enzyme with molecular weight of 25 kDa shows all characteristics of phenol hydroxylases i.e. hydroxylation of phenol to catechol (confirmed by HPLC), substrate dependent NADPH oxidation, absorption spectrum typical of flavoproteins and peptide mass fingerprint corresponding to flavoprotein hydroxylase. The enzyme utilizes phenol with apparent Michealis constant (Km) of 1.71 µM, maximal velocity (Vmax) of 0.4 µM/min with optimal activity at pH 7 and 35°C. Fe(2+)chelators (Phenanthroline and sodium arsenate), heavy metals, denaturants and oxidizing agents showed inhibitory effect on phenol hydroxylation activity of the enzyme. The enzyme has broad substrate specificity against isomeric diphenols, isomeric methylphenols, halogen substituted phenols, amino substituted phenols, nitrophenols, hydroxybenzaldehyde and hydroxylbenzoic acid. The enzyme shows remarkable storage stability at room temperature and at 4 (o)C. The multisubstrate specificity coupled to remarkable storage stability of the microalgal phenol hydroxylase opens up avenues for its application in remediation of a wide range of phenolics released in industrial wastewater or phenolic biosensor application.