To count the cells precisely by using a microfluidic cytometer, a three-dimensional focusing microfluidic chip was designed by UV-photolithography based on SU-8 photoresist. The Polydimethyl Siloxane (PDMS) was used to cast doubly to replicate its structure and to shorten the fabrication cycle of microfluidic chip and reduce its costs. First, the PDMS was taken to fabricate a mold with a three-dimensional focusing microfluidic channel by the oblique exposure lithography of SU-8 photoresist and immersion lithography. Then, the PDMS was used to cast firstly for the SU-8 microfluidic to obtain a PDMS negative mode structure. After the negative mode structure was treated, it was casted again and the PDMS microfluidic detection channel which was consistent with that of the original mold was achieved. Finally, the PDMS microfluidic channel with a cover plate was packaged by using a certain packaging method and the microfluidic chip was obtained. Furthermore, the channel focus effect was tested. The test result shows that the focused width of sample flow changes with the different velocity ratios of sheath flow and sample flow. When the ratio of sheath flow and sample flow is 20∶1, the focused width is about 10.4 μm. The results demonstrate that the chip with the microstructure is reliable, and it meets the requirements of further fluid focus testing. The manufactured microfluidic chip by using this process has a short cycle, low costs and a high efficiency.