Self-assembly of pi-conjugated polymers upon slow precipitation was comprehensively studied by suffusion of a vapor of non-solvents into polymer solutions. However, it was reported that only copolymers with steric hindrance in their backbone tended to form ordered spheres, while homopolymers having a single monomer component were hard to form ordered microstructure. This article presented here a strategy for the self-assembly of microspheres of a pi-conjugated polymer having a single monomer component, e.g., poly(9,9-dihexylfluorene) (PDHF). The microspheres of PDHF were fabricated in the methanol vapor by the non-solvent vapor method. To obtain the optimized assembling parameters, the effect of self-assembling conditions including polymer concentration, the injection volume, the types of the non-solvent vapor, and solvent on the microstructural formation of PDHF were well investigated. The experimental results indicated that the increase of polymer concentration and injection volume led to the increase of partial aggregate of the spheres. Besides, the relatively uniform microsphere could be easily obtained with the concentration ranged from 2.5 to 5.0 mg/mL, and the injection volume of 5.0 mu L in the methanol vapor. Furthermore, the polar solvent was advantageous to form well-ordered microspheres in the methanol vapor.