The use of supported Co-based catalysts is widespread in various catalytic reactions due to their unique structures. The structural sensitivity of these catalysts is closely linked to their particle size and crystal form. Consequently, comprehending the structure–activity relationship requires the development of well-defined Co-based catalysts. Herein, we employed a colloidal wet chemical process and a heterogeneous nucleation method to prepare well-defined Co-based catalysts supported by inert carbon nanospheres. The nanospheres’ surface possesses abundant functional groups that efficiently capture metal complexes and facilitate the nucleation and growth of CoO nanoparticles. By adjusting the Co source concentration, solvent molar ratio, and nucleation growth kinetics, we successfully prepared CoO/carbon sphere (CS) catalysts with different particle sizes and crystal forms. The influence of metallic face-centered cubic (fcc)-Co⁰ particle size in the range of 6.6–17.6 nm on the performance of Fischer–Tropsch synthesis (FTS) using well-defined CoO/CS catalysts has been investigated. The result demonstrated that the turnover frequency (TOF) remained constant for CoO/CS catalysts with metallic fcc-Co⁰ particle size larger than 7.7 nm. However, both the selectivity and the activity changed for CoO/CS catalysts with smaller particles (< 7.7 nm). Significantly, when metallic fcc-Co⁰ particle size was reduced from 17.6 to 7.7 nm, the cobalt time yield increased to 6.7 μmol_CO·g_Co⁻¹·s⁻¹, indicating improved catalytic activity. At the same time, the CH₄ selectivity decreased to 4.9%, suggesting a higher preference for hydrocarbon production. These findings demonstrate the importance of particle size in Co catalyzed Fischer–Tropsch synthesis. The use of well-defined CoO/CS catalysts offers valuable insights into the structure–activity relationship, leading to a better understanding of Co catalyzed Fischer–Tropsch synthesis.