The invention discloses a generation method of a dynamic binary code test case. The generation method comprises the following steps: a dynamic binary insertion tool is achieved to collect the context information including a register, a memory, a thread, system invoking, base loading and the like in the execution process of the binary code, transmitting such information into a binary code translation assembly, converting the information into an intermediate language, carrying out symbol execution on the intermediate language, disseminating symbol input, and collecting a path constraint depending on the input. And then, inputting the path constraint into a path generation assembly, providing a new in-step branch-path mutation algorithm and a strategy, generating new path constraints as much as possible after each symbol execution, using a theorem prover to solve the new path constraint, obtaining the new input satisfying the path constraint, transmitting the new input to the binary code to carry out specific execution for one time, obtaining new path constraints from the new execution in continuously circular manner, generating new inputs, and improving the cover ratio of the binary code.