This study assessed the effect of the carbon/nitrogen (C/N) ratio on the hydrogen production from sucrose-based synthetic wastewater in upflow fixed-bed anaerobic reactors. C/N ratios of 40, 90, 140, and 190 (g C/g N) were studied using sucrose and urea as the carbon and nitrogen sources, respectively. An optimum hydrogen yield of 3.5 mol H2 mol−1 sucrose was obtained for a C/N ratio of 137 by means of mathematical adjustment. For all C/N ratios, the sucrose removal efficiency reached values greater than 80% and was stable after the transient stage. However, biogas production was not stable at all C/N ratios as a consequence of the continuous decreasing of the specific organic loading rate (SOLR) when the biomass accumulated in the fixed-bed, causing the proliferation of H2-consuming microorganisms. It was found that the application of a constant SOLR of 6.0 g sucrose g−1 VSS d−1 stabilized the system.