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Biocompatible nanogels are highly in demand and have the potential to be used in various applications, e.g., for the encapsulation of sensitive biomacromolecules. In the present study, we have developed water-in-oil microemulsions of sodium alginate sol/hexane/Span 20 as a template for controlled synthesis of alginate nanogels, cross-linked with 3d transition metal cations (Mn2+, Fe3+, and Co2+). The results suggest that the stable template of 110 nm dimensions can be obtained by microemulsion technique using Span 20 at concentrations of 10mM and above, showing a zeta potential of −57.3 mV. A comparison of the effects of the cross-links on the morphology, surface charge, protein (urease enzyme) encapsulation properties, and stability of the resulting nanogels were studied. Alginate nanogels, cross-linked with Mn2+, Fe3+, or Co2+ did not show any gradation in the hydrodynamic diameter. The shape of alginate nanogels, cross-linked with Mn2+ or Co2+, were spherical; whereas, nanogels cross-linked with Fe3+ (Fe–alginate) were non-spherical and rice-shaped. The zeta potential, enzyme loading efficiency, and enzyme activity of Fe–alginate was the highest among all the nanogels studied. It was found that the morphology of particles influenced the percent immobilization, loading capacity, and loading efficiency of encapsulated enzymes. These particles are promising candidates for biosensing and efficient drug delivery due to their relatively high loading capacity, biocompatibility, easy fabrication, and easy handling.