A series of fatty acid quaternary ammonium surfactants (FAQAS 1 a–1 f) derived from renewable resource of fatty acid have been rationally designed and investigated as levelers applied in copper electroplating. Surface tension and contact angle indicated the FAQAS's surfactivity and wettability were closely related to the length of hydrophobic chains. FAQAS 1 a–1 f displayed the capability to inhibit the copper deposition in CV tests and their inhibiting abilities agreed with the order of their surfactivity and wettability. Compound 1 e showed the best inhibition capability, and was comprehensively tested by field‐emission scanning electron microscopy (FE‐SEM), X‐ray photoelectron spectroscopy (XPS) and other electrochemical measurements. The strong inhibition ability of compound 1 e on copper electrodeposition could be attributed to its fast and strong absorption of 1 e on the cathode surface. Moreover, galvanostatic measurements (GMs) indicated that compound 1 e not only cause polarization but also has stable synergistic effects with other additives to form a convection dependent competitive adsorption layer. In addition, through‐hole (TH) electroplating experiments verified that uniform TH could be obtained on copper electrodeposition using the bath containing compound 1 e. This work may shed some light on the pratical application of surfactants as effective leveler in acid copper electroplating.