Despite serving as a gold standard for protein analysis, the classic enzyme-linked immunosorbent assay (ELISA) is currently challenged by the ever-increasing needs of sensitivity and simplicity. Towards the ongoing needs, recent advances in nanomaterials have offered numerous promising tools for enhancing the performance and broadening the applicability of ELISA. In this review, we highlight nanomaterial-enabled strategies that drastically improve the assay performance without significantly altering the classic ELISA format. Particular attention will be focused on the functional roles of nanomaterials as novel readout systems in ELISA, including those serving as substrate-alternatives, enzyme-alternatives, and non-enzymatic signal amplifiers.