Selenium-containing polymers are a new type of responsive polymer material. Here, a selenium-containing amphiphilic block copolymer (PEG-PUSe-PEG) has been investigated using atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS). The deviation between force-extension curves of PEG-PUSe-PEG in water and in DMSO is found to be related to the disassembly of the micellar structures in water. SMFS experiments on PEG-PUSeox-PEG suggest that the change from selenide to oxidized selenone contributes significantly to the change in amphiphilicity, without obviously influencing the single-chain elasticity.