Single-molecule detection enables direct characterization of annealing/melting kinetics of nucleic acids without the need for synchronization of molecular states, but the current experiments are not carried out in a native cellular context. Here we describe an integrated 3D single-molecule tracking and lifetime measurement method that can follow individual DNA molecules diffusing inside a mammalian cell and observe multiple annealing and melting events on the same molecules. By comparing the hybridization kinetics of the same DNA strand in vitro, we found the association constants can be 13- to 163-fold higher in the molecular crowding cellular environment.