Near-infrared (NIR) fluorescent metal nanodots may have significant advantages in biological detection and bioimaging. Herein, we introduce tunable near-infrared fluorescent gold nanodots (AuNDs) protected by branched polyethylenimine (PEI) modified by surface segmental attachment of sulfhydryl groups (PEI-SH), abbreviated as PEI-SH-AuNDs, for simultaneous gene delivery and cell imaging. The modified PEI endows the resultant PEI-SH-AuNDs with the following excellent advantages. Sulfhydryl groups of PEI-SH anchor to the surface of AuNDs, and such polycations with amine groups give PEI-SH-AuNDs remarkable stability. The cationic polymer PEI-SH with positive charges enables PEI-SH-AuNDs to perform gene delivery, and the gene transfection efficiency can reach 22.8%. Moreover, the fluorescence of PEI-SH-AuNDs is tunable from visible red light (wavelength 609 nm) to NIR light (wavelength 811 nm) via an increase in the size of AuNDs. PEI-SH-AuNDs yielded gene transfection efficiency similar to that of commercial PEI, but showed much lower cytotoxicity and much greater red-shift fluorescence. With excellent photoluminescent properties, such multifunctional fluorescent PEI-SH-AuNDs hold promise in applications to bioimaging and as ideal fluorescent probes for tracking gene transfection behavior.