The invention provides a microfluidic chip for analysis of an in-vitro thrombolytic drug effect and a preparation method. The chip is prepared from elastic polydimethylsiloxane as a material with a molding method, adopts a two-layer structure and comprises a main pipe, four liquid inlets and two micro valves. The drug thrombolysis effect can be analyzed with only a trace quantity of blood and thrombolytic drugs through simulation of the actual state of thrombus clots in a human blood vessel in the main pipe of the chip. The chip has characteristics of simple structure, low preparation cost, little sample consumption and high detection precision, and is applicable to analysis of the thrombolytic effect of the thrombolytic drugs and screening of the drugs.