We present an effective and low-cost method for fabricating microfluidic chip based on excimer laser direct-writing ablation and replica molding. It is based on a newly excimer laser micromachining technique that can accurately machine microfluidic chip microstructure with smooth surface profile. Microfluidic chips with precise and smooth surface profiles are ablated by direct laser machining on an epoxy glue layer sticking on glass substrates. Laser-machined microfluidic chips are replicated by electroforming to obtain inverse metal molds. Finally, polycarbonate (PC) microfluidic chips are replicated from these metal molds using injection molding method. The relation between the process parameters (the translational speed of working platform and the laser fluence) and the micromachining quality (the depth and surface of the microchannel) is investigated. The profile accuracy and surface roughness of the produced microfluidic chip at each stage are measured and monitored. The average upper surface profile accuracy is better than 2 μm and the average surface roughness is less than 20 nm. Experimental data show the controllability and accuracy of this micromachining process. Experimental investigation is performed on the flow characteristics of water in two different roughness rectangle microchannels. The flow characteristics of water in the microchannels fabricated with the proposed combination method are better than those in the microchannels fabricated with laser direct-writing when the flow velocity is small.