[Background] Carbamoyl phosphate is an important precursor in metabolism of arginine and pyrimidine, and plays a key role in biosynthesis of arginine and pyrimidine and its derivatives in microorganisms. [Objective] The synthesis of carbamoyl phosphate through different pathways was investigated and compared in Escherichia coli BW25113. [Methods] Ornithine carbamoyltransferase (OTC) was overexpressed in E. coli BW25113 as a carbamoyl phosphate detecting system. Then carbamate kinase (CK) from different species or carbamoyl phosphate synthase II (CPS II) was overexpressed in E. coli BW25113 to enhance carbamoyl phosphate biosynthesis. These two carbamoyl phosphate biosynthetic pathways (CK pathway and CPS II pathway) were then optimized by adjusting substrates supply and introducing L-glutamine synthetase. [Results] A carbamoyl phosphate detection system was established in vivo by overexpressing OTC in E. coli. By overexpressing CKs from different species in E. coli with OTC, the carbamoyl phosphate biosynthetic pathway was enhanced, and the results showed that CK from E. coli was better than the other two, which produced 2.95±0.15 mmol/L L-citrulline in 9 h whole-cell bioconversion. By overexpressing CPS II in E. coli with OTC, the carbamoyl phosphate biosynthesis was improved, and 3.16±0.29 mmol/L L-citrulline was obtained in the whole-cell biocatalytic process. The CK and CPS II pathways were optimized by changing the concentration of substrate NH4HCO3 to 100 mmol/L and introducing exogenous L-glutamine synthetase (GS). As a result, 4.67±0.55 mmol/L and 6.12±0.38 mmol/L L-citrulline was achieved by strains with CK or CPS II pathway respectively. After the optimization of CPS II pathway with overexpressing GS, extra addition of L-glutamine was not required in the reaction system. [Conclusion] Biosynthesis of carbamoyl phosphate was improved by constructing and optimizing the CK synthetic pathway or CPS II synthetic pathway. CPS II pathway with introducing GS is better than that of the CK synthetic pathway in respect of carbamoyl phosphate synthesis, and provides a more efficient strategy for the synthesis of arginine, pyrimidine and its derivatives.