Four ester cationic Gemini surfactants (EDQ‐n, n represents the carbon number in the raw fatty acid of 12, 14, 16 and 18) were synthesized from fatty acid, N, N‐dimethylethanolamine and 1, 4‐dibromobutane by two‐step reaction of esterification and quaternization. Their surface activities at air/water interface were investigated. The application performance such as water solubility, antistatic property, fabric softness, fabric whiteness, biodegradability, and antibacterial activities were tested and compared with traditional quaternary ammonium salt. The results showed that the γcmc increases with increasing the hydrophobic chain length and EDQ‐12 presented the highest surface activity. The diffusion coefficients calculated from the Ward‐Tordai equation indicates the adsorption process is a mixed‐diffusion kinetic adsorption mechanism. The water solubility of EDQ‐n is higher than dioctadecyl dimethyl ammonium chloride (D1821) and ester quaternary ammonium salt (Esterquats). The fabric softness and whiteness properties of EDQ‐16 and EDQ‐18 are similar to D1821 and Esterquats, which can significantly reduce the cotton fabric softness and have no significant difference in whiteness retention. EDQ‐n demonstrated more readily biodegradation(>90 %) than D1821(0 %) after seven days. In addition, EDQ‐12 and EDQ‐14 show excellent antibacterial activities against Staphylococcus aureus and Escherichia coli in comparison with conventional bacteriostatic agent didecyl dimethyl ammonium chloride (DDAC).