An important prerequisite for surface tension self-assembly is to limit droplets to the target area using wettability contrast structure. In this paper, a method for fabricating wettability contrast structure on composite substrate surface using microsecond pulsed laser is presented. This method can control the percentage of superhydrophobic coating removal area on the surface of composite substrate by changing the laser processing parameters, and then regulate the wettability of the processing area. The mathematical model and simulation model of removal area proportion are established, and the influence of processing parameters on removal area proportion and wettability is studied. The results show that the removal area proportion of processing area is inversely proportional to processing speed and scanning line spacing. Different wettability zoning structures are prepared with different processing parameters and their contact angles are measured. The experimental results show that with the increase of machining speed from 100 mm/s to 9 000 mm/s, the distance between scan lines increases from 20 μm to 150 μm, the percentage of removal area in the processing area decreases, and the contact angle gradually increases from below 5 degrees to 127 degrees. As the absolute distance between the substrate surface and the laser focal plane increases from 0 to 3 mm, the contact angle of the machined area increases from below 5 degrees to over 170 degrees. The results of self-assembly experiments on microchips show that the wettability contrast structure prepared by this method can realize droplet limitation and microchips self-assembly. The research results in this paper provide a new idea for manufacturing self-assembly substrate with controllable wettability.