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Polymer porous microspheres with large specific surface areas and good fluidity have promising important applications in the biomedical field. However, controllable fabrication of porous microspheres with precise size, morphology, and pore structure is still a challenge, and phase separation caused by the instability of the emulsion is the main factor affecting the precise preparation of porous microspheres. Herein, a method combining the iso-density emulsion (IDE) template and microfluidics was proposed to realize the controllable preparation of polymer porous microspheres. The IDE exhibited excellent stability with minimal phase separation within 4 h, thus showing potential advantages in the large-scale preparation of porous microspheres. With the IDE template combined microfluidics technique and the use of a customized amphoteric copolymer, PEG-b-polycaprolactone, polycaprolactone (PCL) porous microspheres with porosity higher than 90% were successfully prepared. Afterwards, the main factors, including polymer concentration, water–oil ratio and homogenization time were investigated to regulate the pore structure of microspheres, and microspheres with different pore sizes (1–30 μm) were obtained. PCL porous microspheres exhibited comparable cell viability relative to the control group and good potential as cell microcarriers after surface modification with polydopamine. The modified PCL porous microspheres implanted subcutaneously in rats underwent rapid in vivo degradation and tissue ingrowth. Overall, this study demonstrated an efficient strategy for the precise preparation of porous microspheres and investigated the potential of the as-prepared PCL porous microspheres as cell microcarriers and micro-scaffolds.