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The recent development of single-molecule sensors (SMS), which detect individual targets one at a time, allows determination of ultra-low concentrations of structurally similar compounds from a complex matrix. Protein nanopores are one of the earliest methods able to resolve the signal from a single molecule, and have already been successfully employed in commercial DNA sequencers. The protein nanopore based SMS, however, remains challenging, largely because the quantitative single-molecule analysis requires recording a sufficient number of signals for statistical significance within a reasonable time frame, thus restricting the lower limit of detection. This review aims to critically evaluate the strategies developed in this field over the last two decades. The measurement principle of nanopore SMS is first elucidated, followed by a systematic examination of the eight common protein pores, and a comprehensive assessment of the major types of sensing applications. A particular emphasis is placed on the intrinsic relationship between the size and charge of protein nanopores and their sensing capabilities for different kinds of analytes. Innovative approaches to lift the performance of nanopore SMS are also analyzed in detail, with a prediction at the end of the most promising future applications. [ABSTRACT FROM AUTHOR]