Organocatalyst-mediated acyl transfer reactions hold promise for selective protein labeling in biological milieu. However, they often suffer from off-target reactions and high background signals because of the requirement of high concentrations of substrates. Here, we report a new catalytic protein acylation strategy promoted by the His-tag/NiNTA interaction. The recognition-assisted activation mechanism allows efficient protein labeling even with >10-fold lower substrate concentrations than conventional reactions, thereby enabling highly selective and efficient cell-surface receptor modification in live cells.