Cold shock proteins (Csps) are a class of highly conserved nucleic acid-binding proteins composed of 65-70 amino acids that form a compact β-barrel structure with five antiparallel β-strands. As nucleic acid-binding proteins, Csps play an important role in bacterial response to cold shock, yet their precise working mechanism remains unclear. It is known that DNA hairpin undergoes folding-unfolding transitions at small constant forces. Magnetic tweezers technique offers distinct advantages for such investigations, particularly its capacity for extended-duration constant-force measurements at pico-Newton force levels, making it ideally suited for characterizing the conformational transition dynamics of DNA hairpin at the low forces of several pico-Newton. In this study, we first stretch DNA hairpin from its N- and C-termini using magnetic tweezers. Then, we sequentially introduce Csp buffer solutions with increasing concentrations into the flow chamber and measure the folding and unfolding rates of the DNA hairpin at different Csp concentrations. We find that within a certain concentration range, increasing Csp concentration significantly reduces the DNA hairpin folding rate while leaving the unfolding rate virtually unchanged. This behavior arises because Csp exclusively binds to single-stranded DNA (ssDNA), interacting with the ssDNA regions of the unfolded DNA hairpin and thereby hindering the folding process. As Csp does not interact with double-stranded DNA (dsDNA), it has negligible effect on the unfolding process. Furthermore, the critical force of DNA hairpin progressively decreased with elevated Csp concentration, demonstrating that Csp effectively destabilizes the hairpin structure. When the Csp concentration reaches sufficiently high levels, we also detect a notable increase in the DNA hairpin's unfolding rate. This phenomenon likely arises from Csp's rapid binding to the newly exposed ssDNA regions of the partially unfolded DNA Hairpin, which prevents refolding and consequently accelerates the unfolding pathway. In force-jump experiments performed with Csp-containing buffers, the binding preference of Csp for either ssDNA or dsDNA can be directly determined by analyzing whether delayed response of DNA hairpin extension occur. In force-increasing jump experiments, we observe no extension delay during the DNA hairpin unfolding process. In contrast, force-decreasing jump experiments revealed significant extension delay during the folding process. These single-molecule measurements provide direct evidence that Csp specifically binds only to ssDNA, and further demonstrate that its binding kinetics occur with remarkable rapidity. This study provides insights to the molecular mechanism of Csps to maintain normal cellular functions in cold chock conditions.