Wastewater treatment and generated biological sludge provide an alternative source of enzymes to conventional industrial production methods. Here, we present a protocol for extracting enzymes from activated sludge using ultrasonication and surfactant treatment. Under optimum conditions, ultrasound disruption of activated sludge gave recovery rates of protease and cellulase enzymes equivalent to 63.1% and ∼100%, respectively. The extracting of enzymes from activated sludge represents a potentially significant, high-value, resource recovery option for biological sludge generated by municipal wastewater treatment.