Proteins are the molecular foundations of life and disease responsible for understanding most biological processes. Nanopore technology devoted to revealing single-molecule behavior has made great breakthroughs for protein identification, detection and analysis, including protein sequencing. Here, we present an overview of the latest advances in protein profiling by nanopores from the identification and quantification of protein biomarkers and protein enzymes to the delineation of protein conformations and interactions at the single-molecule level, focused on the diverse and exciting approaches to protein sequencing. Furthermore, we discuss the primary challenges associated with nanopore-based protein sensing and recommend potential strategies respond to these challenges from the perspective of nanopore engineering and data processing.