Pluronic is a surface-active poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymer. The PEO chains of the triblock form a hydrophilic, protein-repelling interface at hydrophobic surfaces. The protein-repelling property of Pluronic surfactants was used as an activity-preserving foundation on which to develop a scheme for specific protein immobilization at hydrophobic interfaces through immobilized metal affinity. A nitrilotriacetic acid (NTA) group was coupled to the terminal hydroxyl groups of the PEO chains of Pluronic F108 to create a metal-chelating Pluronic (F108-NTA). Recombinant firefly luciferase (FFL) with C-terminal histidine tags was used as a test protein. Histidine-tagged FFL adsorbed strongly to untreated polystyrene beads but retained much less than 1% of its bioluminescence activity. An equivalent amount of histidine-tagged FFL bound to polystyrene beads treated with the chelating Pluronic, but only in the presence of Ni2+ ions. The firefly luciferase immobilized on the chelating Pluronic retained at least 93% of its bioluminescence activity. The results demonstrate that the chelating Pluronic is a simple and versatile reagent for specific, oriented immobilization of histidine-tagged proteins on hydrophobic interfaces.