A thermodynamic equilibrium model of the enzymatic hydrolysis of penicillin G to produce 6-aminopenicillanic acid in a cloud point system, a novel biphasic system for extractive biocatalysis, was developed. An extractive reaction coefficient was proposed to set up the relationship between the effect of the cloud point system to the enzymatic hydrolysis of penicillin G and the operation parameters, such as pH, phase volume ratio, and partitioning coefficients of reactants or products. On the basis of the model analysis, an experimental procedure of a discrete semibatch process was developed and a high product 6-aminopenicillanic acid concentration was achieved without pH control. This envisages an industrial application of the eco-friendly semibatch process with a recycle of the coacervate phase of the cloud point system.