Rennet is a form of enzyme preparation of animal, plant or microbial origin that is used in cheese production to induce milk coagulation. The natural rennet of animal origin was used for centuries until high demand resulted in development and use of microbial and GMO modified rennets in the last century. Natural rennet is produced from abomasum of young suckling calves, lambs, and goats. To increase the stability and long-term activity of the rennet, the process of encapsulating the rennet in alginate microparticle formulations is carried out, which achieves the immobilization and release of enzymes at certain time intervals. Even recently, the stabilization of rennet, especially in the case of accelerated cheese ripening, has been attracting considerable interest. In this work, the optimization of the concentration of sodium alginate and calcium chloride for the encapsulation of rennet was carried out. Under optimal conditions, alginate was 1.5% and CaCl2 was 4%. Under optimal conditions, encapsulation efficiency (EE%), filling capacity (LC), microparticle size, and degree of swelling (Sw) were estimated to be 56.8%, 80%, 1386.51 ± 0.231 mm, and 61.8%, respectively. In addition, the data on rennet (enzyme) release were adapted to the Korsmeyer-Peppas model, and the exponent n > 0.43 indicates that the controlling mechanism of the release is anomalous transport kinetics (combination of two diffusion mechanisms and swelling and relaxation of polymer chains). Electrostatic interactions between the enzyme, alginate, and CaCl2 were confirmed by infrared spectroscopy, and the morphology of the microparticle was determined using an optical microscope (OM) and a scanning electron microscope (SEM).