The mitochondrial DNA helicase TWINKLE, a hexameric ring-shaped helicase, plays a crucial role in maintaining mitochondrial DNA integrity. TWINKLE translocates along one DNA strand, unwinding the duplex by excluding the complementary strand through coordinated ATP hydrolysis. However, the precise mechanisms underlying this process remain incompletely understood. In this study, we utilized single-molecule Förster Resonance Energy Transfer (smFRET) to investigate the mechanisms of TWINKLE-mediated DNA unwinding. Our results reveal that TWINKLE occasionally pauses during unwinding, with the rate of unwinding and the duration of pausing strongly influenced by ATP concentration, but not by the presence of DNA mismatches or mitochondrial single-stranded DNA-binding protein (mtSSB). These findings suggest that the pausing events primarily arise from stochastic ATP hydrolysis within the helicase subunits. DNA mismatches exacerbate TWINKLE's pausing and dissociation from DNA, thereby impairing DNA unwinding. In contrast, mtSSB significantly mitigates helicase dissociation by stabilizing TWINKLE-DNA interactions. This study provides novel insights into the functional dynamics of TWINKLE, highlighting the role of ATP hydrolysis in orchestrating single-stranded DNA translocation, the detrimental effects of DNA mismatches on DNA unwinding, and the critical role of mtSSB in supporting helicase function.