A facile amperometric biosensor that included oxidase mimicking Co/2Fe metal-organic framework (MOF) for sialic acid (SA) detection was prepared. Amperometric SA biosensor was constructed on a gold screen-printed electrode via immobilization of Co/2Fe MOF and N-acetylneuraminic Acid Aldolase (NANA-Aldolase) enzyme, respectively. NANA-Aldolase enzyme converts free SA into pyruvate and N-acetyl-d-mannosamine. After this conversion, oxidase mimicking Co/2Fe bimetallic MOF converts pyruvate into acetylphosphate and O2 into H2O2. Investigation of analytical characteristics resulted with the linear range of 0.02 mM–1.00 mM of SA concentration with limit of detection value of 0.026 mM. Sample application studies with developed SA biosensor were carried out with GD3 ganglioside and HeLa cancer cell lines which have high SA concentrations while A549 cell lines were also used as control group. Before detecting free SA, the bound SA was freed from SA sources where every step was monitored via electron impedance spectroscopy. Then, free SA was successfully detected with the amperometric SA biosensor and as a result, more practical and accurate system was developed. Display omitted •An amperometric sialic acid (SA) biosensor based on oxidase mimicking Co/2Fe metal-MOF was prepared.•The biosensor included Co/2Fe MOF and NANA-Aldolase enzyme immobilized on AuSPE.•MOF converts pyruvate into acetylphosphate and oxygen into H2O2.•With developed biosensor, free SA was successfully detected.•Before detection of free SA, bound SA was released.