Handling molecules one at a time and measuring signals from the single molecules provides a unique opportunity to address fundamental biological problems and technical challenges as well. In this seminar, I will talk about our two recent experiments that cannot be paralleled in conventional bulk measurements. First, by combining two orthogonal techniques of single-molecule FRET and magnetic tweezers, we reveal how the 20S particle consisting of NSF and .α-SNAP disassembles single SNARE complexes. We observed that NSF exploits only one round of ATP binding and hydrolysis to disassemble the SNARE complex, one of the most stable protein complexes, suggesting an incredibly high mechanical efficiency of NSF compared to other AAA+ ATPases. Second, by employing our real-time single-molecule fluorescence imaging as the detection scheme, we have recently demonstrated a single-molecule version of the co-immunoprecipitation (co-IP) analysis, which provides an improvement of five orders of magnitude in the time resolution. With the single-molecule sensitivity and millisecond time resolution, it is possible to determine the signaling kinetics of native proteins and detect changes in the protein-protein interactions in a given tumour tissue. This will shed light onto the molecular lesions that drive individual cancers at the level of protein-protein interactions. I will close the talk with brief outlook for future researches.