Human serum albumin (HSA) levels in the body are crucial for the accurate diagnosis of various health issues, necessitating continuous monitoring and novel detection methods. This study focused on the development and synthesis of two novel D-π-A small molecule near-infrared fluorescent probes, PTPA-B and CPTPA-B. The probes demonstrated selective detection of HSA with a rapid response time of 1 min and high sensitivity, featuring limits of detection (LOD) of 0.102 μM and 1.702 μM. Under physiological conditions, the intrinsic fluorescence of these probes is minimal due to intramolecular charge transfer (TICT). Upon entering the hydrophobic cavity of HSA, the probes exhibit red fluorescence by inhibiting the TICT process. Additionally, the probes exhibited enhanced biocompatibility in living cells and enabled fluorescence imaging of L-02 live cells. Therefore, the PTPA-B fluorescent probe serves as an effective tool for serum detection, revealing its potential applications in live cell imaging and precise clinical diagnosis.