The invention relates to real-time data acquisition system and method of a high-resolution imaging instrument, used in a data acquisition card capable of acquiring data at a high speed and a computer capable of storing data at a high speed. The data acquisition card comprises an interface unit, a buffer unit, a data sending unit and a control unit, and the interface unit is connected with an imaging instrument and can convert an LVDS (Low Voltage Differential Signaling) signal into a TTL (Transistor-Transistor Logic) level signal. The invention solves the technical problems that a traditional image acquisition and storage system can not satisfy the real-time acquisition and storage requirements of high-resolution imaging data. The invention has the advantages of small use size, portability, simplicity, convenience, stable working performance, reliability, and the like.