We describe a new method to measure the activity of protein in miniscule quantities using a carbon nanotube nanoneedle interface. The unique features of this new method are: (a) immobilization of a few molecules on the interface; (b) subsequent translocation and isolation of them near the tip of a position-actuated nanoneedle; and (c) fixed, defined and unhindered molecular position to allow rapid and real time sensing and monitoring. The kinetic bioactivity of immobilized alkaline phosphatase (AP) molecules was measured as test model. Results show no decrease in enzymatic activity compared to the solution phase enzyme reaction, suggesting that the immobilization provided unhindered access for ligand binding and minimal conformational modulation caused by undesired surface interactions.