Using aldehyde-functionalized Pluronic as the reactive surfactant, enzyme-Pluronic conjugates with sizes ranging from nanometers to micrometers were synthesized in reverse emulsions. Compared with the direct conjugation in aqueous solution, this method gave an increased conjugation efficiency and well-controlled size of the conjugates. The versatility of this method was validated using horseradish peroxidase (HRP), Candida rugosa lipase (CRL) and Candida antarctica lipase B (CALB). The resulting enzyme-Pluronic conjugates showed greatly enhanced apparent activity compared to free enzymes in organic media.