Many important biomarkers for devastating diseases and biochemical processes are proteins present at ultralow levels. Traditional techniques, such as enzyme-linked immunosorbent assays (ELISA), mass spectrometry, and protein microarrays, are often not sensitive enough to detect proteins with concentrations below the picomolar level, thus requiring the development of analytical techniques with ultrahigh sensitivities. In this review, we highlight the recent advances in developing novel techniques, sensors, and assays for ultrasensitive protein analysis. Particular attention will be focused on three classes of signal generation and/or amplification mechanisms, including the uses of nanomaterials, nucleic acids, and digital platforms.