Gold nanorod (GNR) was a promising bioimaging probe in the third optical tissue window (1600–1800 nm). In this paper, hydrophilic GNRs with proper dimensions were synthe- sized according to the classical seed mediated method. Their nanostructures were taken with a transmission electron microscope and their extinction spectra were recorded on a UV-vis scanning spectrophotometer. Their nonlinear optical features were recorded by a home built measurement system, under the femtosecond (fs) laser excitation of 1580 nm, which was selected as the demon- stration of the third optical tissue window. A sharp third harmonic generation (THG) signal and a mild three photon luminescence (3PL) signal could be clearly distinguished. These signals could be utilized for multichannel nonlinear bioimaging in the third optical tissue window. Negatively charged polystyrene sulfonate (PSS) and positively charged polyallylamine hydrochlo- ride (PAH) were separately coated onto the positively charged GNRs through electrostatic adher- ence. The PAH-PSS coated GNRs were added in the culture medium of HeLa cells. Multichannel nonlinear optical imaging of cancer cells stained with GNRs was performed with a 1580 nm fs laser coupled scanning microscope. The signals were filtered by a 665 nm dichroic mirror, and then splitted by another 560 nm dichroic mirror into the green and red imaging channels. HeLa cells can be clearly visualized in both green and red channels with good contrast. For HeLa cells without GNRs treatment, there were no such signals detected in these channels. This was a pioneer work of the multichannel nonlinear bioimaging in the third optical tissue window with GNRs and would be helpful for our further studies on in vivo bioimaging in this window with deeper penetration.