Proteins are responsible for the occurrence and treatment of many diseases, and therefore protein sequencing will revolutionize proteomics and clinical diagnostics. Biological nanopore approach has proved successful for single‐molecule DNA sequencing, which resolves the identities of 4 natural deoxyribonucleotides based on the current blockages and duration times of their translocations across the nanopore confinement. However, open challenges still remain for biological nanopores to sequentially identify each amino acid (AA) of single proteins due to the inherent complexity of 20 proteinogenic AAs in charges, volumes, hydrophobicity and structures. Herein, we focus on recent exciting advances in biological nanopores for single‐molecule protein sequencing (SMPS) from native protein unfolding, control of peptide translocation, AA identification to applications in disease detection. Nanopore electrochemistry offers a bright prospect for single‐molecule protein sequencing by measuring specific interactions between amino acids based on their natural structure and chemistry continuity and diversity. This Minireview focusses on recent advances in biological nanopores from protein unfolding, peptide translocation, amino acid identification to diagnostic application.