Rabies virus (RV) is the most widely used vector for mapping neural circuits. Previous studies have shown that the RV glycoprotein can be a target to improve the retrograde transsynaptic tracing efficiency. However, the current versions still label only a small portion of all presynaptic neurons. Here, we reshuffled the oG sequence, a chimeric glycoprotein, with positive codon pair bias score (CPBS) based on bioinformatic analysis of mouse codon pair bias, generating ooG, a further optimized glycoprotein. Our experimental data reveal that the ooG has a higher expression level than the oG in vivo, which significantly increases the tracing efficiency by up to 12.6 and 62.1-fold compared to oG and B19G, respectively. The new tool can be used for labeling neural circuits Therefore, the approach reported here provides a convenient, efficient and universal strategy to improve protein expression for various application scenarios such as trans-synaptic tracing efficiency, cell engineering, and vaccine and oncolytic virus designs.