Petite integration frequency 1 (PIF1) helicases are ubiquitous enzymes which play vital roles in nearly all DNA metabolic processes. In recent years, the biochemical activity and three-dimensional structure of several PIF1 helicases have been reported, but there are few reports on the PIF1 helicase of bacteria living in extreme environments. In this paper, a series of biochemical and biophysical techniques were used to study the Thermodesulfovibrio yellowstonii PIF1 (Ty.PIF1) helicase in many aspects. Ty. PIF1 was obtained with a purity of over 90% and good uniformity using the prokaryotic expression and purification system. Ty.PIF1 is a monomer with a calculated molecular weight of 60 kD in solution. Ty. PIF1 has high thermal stability. The secondary structure remains stable when the temperature is below 65 ℃, and the secondary structure changes only when the temperature is above 70 ℃. The optimal unwinding temperature of Ty.PIF1 in vitro is 45 ℃, which is not the optimal temperature for the survival of thermodesulfovibrio yellowstonii. It indicates that when Ty.PIF1 exerts its enzymatic activity in vivo, it may require the participation of other cofactors. Ty.PIF1 can exert unwinding activity in a wide temperature range (20-55 ℃), and the presence of enzyme activity at 55 ℃ indicates that Ty.PIF1 has heat-resistant properties. Ty.PIF1 prefers to bind to substrates containing ssDNA, but there is certain requirement for the length of the ssDNA, which is at least 4 nt in length. Ty.PIF1 can also bind to the G