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Monodispersed nickel (Ni) and cobalt (Co) nanoparticles (NPs) with different sizes are synthesized via the thermal decomposition of organometallic precursors by controlling the reaction temperature and surfactant amount. X-ray diffraction analysis of the as-prepared NP samples shows the formation of cubic Ni metal phases with good crystallinity, while the cubic Co metal samples are semi-amorphous. Transmission electron microscopy characterization further confirms that two Ni NP samples with average sizes of 9 and 27 nm, and Co NPs with an average size of 6 nm are successfully prepared with a narrow size distribution. Furthermore, catalytic performance of these monodispersed NPs towards the hydrodesulfurization (HDS) reaction, which plays a pivotal role in the upgrading of heavy crude oil, is evaluated under reservoir-relevant conditions using thiophene as a sulfur-containing model compound. Different parameters including particle size, catalyst dosage, hydrogen donor ratio, temperature, and reaction duration are systematically studied to optimize the catalytic HDS performance. The morphology and size of the spent NP catalysts after the reaction are also analyzed. The results show that the 9 nm Ni NPs exhibit the best HDS activity and stability compared with other catalysts, which suggests that such well-dispersed Ni NPs are promising candidates for the in-situ upgrading and recovery of heavy crude oil from underground reservoirs.