Selective localization of biomolecules at the hot spots of a plasmonic nanoparticle is an attractive strategy to exploit the light–matter interaction due to the high field concentration. Current approaches for hot spot targeting are time-consuming and involve prior knowledge of the hot spots. Multiphoton plasmonic lithography is employed to rapidly immobilize bovine serum albumin (BSA) hydrogel at the hot spot tips of a single gold nanotriangle (AuNT). Regioselectivity and quantity control by manipulating the polarization and intensity of the incident laser are also established. Single AuNTs are tracked using dark-field scattering spectroscopy and scanning electron microscopy to characterize the regioselective process. Fluorescence lifetime measurements further confirm BSA immobilization on the AuNTs. Here, the AuNT-BSA hydrogel complexes, in conjunction with single-particle optical monitoring, can act as a framework for understanding light–molecule interactions at the subnanoparticle level and has potential applications in biophotonics, nanomedicine, and life sciences.