Laser micro/nanomachining technology for soft matter achieves the purpose of fabricating the spherical structures of soft matter by combing laser-assisted mechanical injection and controllable self-assembly, which has significant advantages in comparison with conventional methods like droplet microfluidics. In this study, the effects of laser parameters such as laser energy, beam size, and irradiation position on the injection are investigated. It is found that there also exists one upper limit of the laser energy, and if the laser irradiation is too strong, it can introduce a convection flow of liquid crystal rather than trigger off the injection of guest microdroplets. Thus, the laser injection can be achieved in a specific energy range of the laser irradiation. By manipulating the laser beam with a smaller size, the guest water microdroplets can be injected at the preselected location on the surface of a host liquid crystal droplet. In addition, the influences of material parameters such as the surfactant concentration, the material type and phase state of liquid crystal on the laser-assisted mechanical injection, and the size of the injected guest droplet are investigated. It is found that the liquid crystal droplet with higher surfactant concentration requires less energy from the laser irradiation to generate enough mechanical force to trigger off the injection. Because under the same temperature increment, the liquid crystal droplet with higher ion concentration enjoys a stronger surface tension gradient. By comparing several different types of liquid crystals, it is found the injection of guest droplets into a host with a higher elastic constant liquid crystal can be more difficult. The influences of the material type of liquid crystal and the concentration of surfactant on the critical size of guest microdroplets are summarized. Finally, the defect lines of liquid crystal are introduced as the self-assembly template, through which microdroplets of liquid crystal with the sophisticated spherical structure are fabricated. The self-assembly kinetic behaviors of guest droplets in the defect line are analyzed. The laser micro/nanomachining technology of soft matter can be applied to the extreme processing and application development of 3D spherical structures in the fields of optoelectronics, photonics, and biomedicine.