Glucose biosensors based on porous material of alginate cryogel has been developed, and the cryogel provides a large surface area for enzyme immobilization. The alginate cryogel has been supplemented with NiFe2O4 nanoparticles to improve the electron transfer for electrochemical detection. The fabrication parameters and operational conditions for the biosensor have also been optimized. The results showed that the optimum addition of NiFe2O4 nanoparticles to the alginate solution was 0.03 g/mL. The optimum operational conditions for the electrochemical detection were a cyclic voltammetry scan rate of 0.11 V/s, buffer pH of 7.0, and buffer concentration of 150 mM. The fabricated alginate NiFe2O4 nanoparticles cryogel-based glucose biosensor showed a linear response for glucose determination with a regression line of y = 18.18x + 455.28 and R² = 0.98. Furthermore, the calculated detection limit was 0.32 mM and the limit of quantification was 1.06 mM.