We describe a new method for measuring the activity of protein in miniscule quantities using a carbon nanotube nanoneedle. The unique features of this new method are (a) the immobilization of a few molecules; (b) subsequent translocation and isolation of them near the tip of a position-actuated nanoneedle, and (c) a fixed, defined, and unhindered molecular position to allow rapid 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 that of the solution-phase enzyme reaction, suggesting that the immobilization provided unhindered access for ligand binding and minimal conformational modulation caused by undesired surface interactions.