The prompt introduction of single-molecule science into undergraduates’ curricula is an urgent but challenging task. However, the research experiments are often too complicated and the instruments are too expensive to be widely transferred into a compulsory course of undergraduate students. Herein, we introduce a recently developed nanopore technique into an accessible laboratory course for students with the advantages of simple operation, high repeatability, and low cost. The nanopore course serves to help undergraduates in practice to understand single-molecule behaviors, think at a single molecule level, and gradually develop single-molecule solutions for scientific issues. The learning activities for undergraduate students include setup of the instrument, fabrication of a biological nanopore on an artificial lipid bilayer membrane, detection of individual oligonucleotides, and data analysis. An experiment example is single-molecule detection of the model DNA sample poly­(dA) n (n = 4, 5) using the aerolysin nanopore. As our developed nanopore sensing system is simple, portable, and easy to handle, the experiment course could be implemented and popularized in undergraduates’ laboratory education. For the undergraduate students who have taken general chemistry and/or biochemistry courses, this hands-on experience of nanopore experiments would benefit them with a better understanding of the chemical world at a single-molecule level and further using single-molecule tools in various applications including DNA and/or protein sequencing.