Although a plethora of nonviral gene vectors have been developed for potential gene therapy, imageable gemini surfactants with stimuli-responsiveness and high transfection efficiency are still scarce for gene delivery. Herein, three gemini amphiphiles (DEDPP-4/8/12) consisting of a aggregation-induced emission (AIE) central fluorophore: 5,6-diphenylpyrazine-2,3-diester (DEDPP), decorated with triazole-[12]aneN3 as the hydrophilic moiety and alkyl chains of various lengths as the hydrophobic moiety, were designed and synthesized for trackable gene delivery via optical imaging. All three amphiphiles exhibited ultralow critical micelle concentrations (CMCs) (up to 3.40 × 10−6 M), prominent two-photon absorption properties and solvatochromic fluorescence. Gel electrophoresis assays demonstrated that the migration of plasmid DNA was completely retarded after condensation with these gemini amphiphiles at low concentrations (up to 10 M). In addition, the ester bond in these amphiphiles may facilitate vector degradation and DNA release, in response to esterase and acidic environment inside cells. Upon self-assembly with DOPE to form liposomes, DEDPP-8/DOPE achieved the best transfection efficiency in four cell lines, and the transfection efficiency of DEDPP-8/DOPE in HeLa cell lines was 23.5-fold higher than that of Lipo2000, which is unusually high for small organic molecules based nonviral vectors. Furthermore, excellent transfection efficiency of DEDPP-8/DOPE was obtained in the presence of serum, and red fluorescence protein (RFP) gene was successfully transfected in zebrafish embryos. Both one- and two-photon fluorescence imaging clearly demonstrated the delivery process of plasmid DNA. This study demonstrated that gemini-type amphiphiles composed of a two-photon fluorophore core conjugated with triazole-[12]aneN3 via an ester bond afforded an unprecedentedly high transfection efficiency with excellent bio-compatibility, which may provide new insights for the design and development of multifunctional nonviral gene vectors for imageable gene delivery.