Photoresponsive uniform colloidal spheres were constructed from an amphiphilic azobenzene-containing random copolymer, poly {2-[4-(phenylazo)phenoxy] ethyl acrylate-co-acrylic acid} (PPAPE), and were characterized by using TEM, SEM, AFM, SLS, and DLS measurements. The photoisomerization of the azobenzene units was studied and used as a tool to probe the structure variation during the sphere formation process. The spheres were formed through gradual hydrophobic aggregation of the polymeric chains in THF-H2O dispersion media, which was induced by a steady increase in the water content. The polymeric chains started to aggregate and form the colloidal spheres at the critical water content (CWC). CWC was related with the initial concentration of the polymer in the THF solution and estimated to be 25% (vol %) when the initial concentration was 1.0 mg/mL. The colloids grew as the water content increased above the CWC. After the water content reached 40-50% (vol %), both the trans-to-cis photoisomerization rate and the isomerization degree at the photostationary state decreased significantly as the water content increased. It indicated that the isomerization was hindered by the polymeric chain collapse and entanglement occurring at this stage of the sphere growth process. Upon UV light irradiation, the photoinduced deaggregation behavior of the spheres was controlled by the water contents of the dispersion media. The understanding obtained from above observations can give some insight into the mechanism for the formation of the uniform colloidal spheres from polydispersed random copolymers and can be used to control the colloidal structures by adjusting the preparation conditions.