To make the gold nanorod (AuNR) a better photoluminescence (PL) probe for cell imaging under two-photon excitation (TPE), the effect of the aspect ratio of AuNRs was studied. The AuNRs with the aspect ratios of 2.7, 3.2, 4.1, and 4.5 and correlated longitudinal surface plasmon resonance (LSPR) bands of 710, 760, 820, and 870 nm were compared. The approach of two-photon excited PL was used to measure the two-photon absorption cross section (TPACS) of these AuNRs in aqueous solutions. Under TPE of an 800-nm femtosecond laser, the TPACS of AuNRs with an aspect ratio of 3.2 was found to be the highest (about 3 × 109 GM), and that of AuNRs (aspect ratio of 2.7) was only 1.5 × 109 GM. The probe function of these two AuNRs was further compared in cell imaging studies using the human liver cancer cell (QGY) as the cell model. Both TPE PL image and confocal reflectance image of AuNR-loaded cells were acquired comparatively in measurements. The brightness and contrast of confocal reflectance images for these two AuNRs in cells are similar. In contrast, the PL images of cellular AuNRs (2.7) under TPE of 800 nm are weak but that of cellular AuNRs (3.2) is much better. These results show that when the LSPR band of AuNRs is coincided with the excitation wavelength, the TPACS of these AuNRs will be enhanced ensuring a good quality of cell imaging under TPE. The LSPR band is correlated to the aspect ratio of AuNRs. Therefore, in cell imaging studies with TPE, the aspect ratio effect of AuNRs should be taken into consideration.