Objective To detect the expression profiles of 14-3-3η mRNA in different polarized macrophages, analyze the bioinformation, and construct the prokaryotic expression vector of 14-3-3η in order to provide an experimental basis for further study on macrophage polarization. Methods After the mRNA of bone marrow derived macrophages from C57BL/6 mice was extracted, real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression of 14-3-3η after reverse transcription. ProtParam tool, SOPMA and other online statistics were used to analyze the amino acid sequence of 14-3-3η. With mouse macrophage cDNA as template, PCR was employed to amplify the target genes, and the products were inserted into pGEX-4T-3 vector. After verification by double-enzymes digestion and sequencing, the correct recombinant plasmids were transformed into the Escherichia coli strain BL21. Then the bacteria were induced by isopropyl β-D-1-thiogalactopyranoside (IPTG), and the expressed proteins were purified and identified by SDS-PAGE and Western blotting. Graphpad prism 5 was used to perform the statistical analysis. Paired t test was employed for the comparison among different samples. Results The mRNA expression level of 14-3-3η was decreased in M1 macrophages, but it was increased in M2 macrophages. Bioinformatic analyses showed that α-helix (62.60%) was the main component of its secondary structure, and it had poor hydrophobicity (hydrophobic coefficient=-0.607). Enzymes digestion and sequencing indicated that the vector pGEX-4T-3-14-3-3η was successfully constructed. GST-14-3-3η protein was induced and identified by SDS-PAGE and Western blotting. Conclusion Primary bioinformatic analyses, successful expression and purification of fusion protein GST-14-3-3η are achieved in this study, which founds the experimental basis for 14-3-3η function in macrophage polarization.