We investigated the direct electrochemistry of glucose oxidase (GOx) at gelatin-multiwalled carbon nanotube (GCNT) modified glassy carbon electrode (GCE). GOx was covalently immobilized onto GCNT modified GCE through the well known glutaraldehyde (GAD) chemistry. The immobilized GOx showed a pair of well-defined reversible redox peaks with a formal potential (E(0)') of -0.40V and a peak to peak separation (ΔE(p)) of 47mV. The surface coverage concentration (Г) of GOx in GCNT/GOx/GAD composite film modified GCE was 3.88×10(-9)mol cm(-2) which indicates the high enzyme loading. The electron transfer rate constant (k(s)) of GOx immobilized onto GCNT was 1.08s(-1) which validates a rapid electron transfer processes. The composite film shows linear response towards 6.30 to 20.09mM glucose. We observed a good sensitivity of 2.47μA mM(-)(1)cm(-2) for glucose at the composite film. The fabricated biosensor displayed two weeks stability. Moreover, it shows no response to 0.5mM of ascorbic acid (AA), uric acid (UA), acetaminophen (AP), pyruvate (PA) and lactate (LA) which shows its potential application in the determination of glucose from human serum samples. The composite film exhibits excellent recovery for glucose in human serum at physiological pH with good practical applicability.