Graphene oxide (GO) membrane, possessing well-aligned laminar interlayer channels and oxygen bearing groups, offers great potentials as proton conductor in fuel cells. Yet, the scarcity and non-uniform distribution of proton conducting sites in the interlayer channels often lead to low conductivity and hinder the use of GO membranes for proton conduction. Single strand deoxyribonucleic acid (ssDNA) molecules bear abundant phosphate and amidogen groups which can act as proton conducting sites. The sequentially arranged phosphate and amidogen groups are in a linear molecule chain, which accords with the ideal arrangement of proton conducting sites. Herein, we intercalated ssDNA into GO interlayer channels via a pre-assembly process to create high-efficient proton conducting channels. GO nanosheets offer well-aligned 2D physical channels and ssDNA provides large amount of sequentially arranged proton conducting sites, which synergistically enhance proton conduction. The DNA@GO-3 membrane shows a proton conductivity of 564.8 mS cm−1 at 80 °C and 98% RH, which is 4.4-fold higher than that of pristine GO membrane and among the highest of the reported GO-based membranes. The H2/O2 single fuel cell performance is improved by nearly 3 folds in terms of maximum power density.