In recent years, researchers have drawn inspiration from natural ion channels to develop various artificial nanopores/nanochannels, including solid-state and biological. Through imitating the precise selectivity and single molecule sensing exhibited by natural ion channels, nanopores/nanochannels have been widely used in many fields, such as analyte detection, gene sequencing and so on. In these applications, the surface functionalization of nanopores/nanochannels directly determines the effectiveness in quantitative analysis and single molecule detection. To explore the modification of different probes on nanopores/nanochannels, this review emphasizes the functionalization of nanopores/nanochannels using small molecules, peptides, nucleic acids, composite molecules and proteins through amidation reactions. In addition, we also present perspectives on the developmental prospects of nanopores, with the goal of enhancing our understanding of nanopore sensing technologies and their functionalization strategies. We have noted that this covalent reaction strategy provides an efficient, versatile and stable modification method for biological and solid-state nanopores/nanochannels.