In this study, a two-photon active DiphenthioER1 was screened from the four pyridinium sulfonate salt derivatives (TriphenER1-2 and DiphenthioER1-2) for imaging endoplasmic reticulum (ER) and tracking the dynamics of the ER morphology. The photophysical properties of TriphenER1-2 and DiphenthioER1-2 were systemically investigated both experimentally and theoretically, revealing that they possess large Stokes shifts, and large two-photon absorption cross-sections from 163 GM to 2023 GM in the near infrared region using a Z-scan method by avoiding spontaneous fluorescence, deep tissue penetration, and low cell damage in living cells. Among them, the DiphenthioER1 compound was found to exhibit the highest cellular uptake ability and two-photon fluorescence signals using confocal microscopy. DiphenthioER1 successfully targeted the ER and induced ER-stress, subsequently nuclear misshaping and mitochondria-mediated apoptosis have been displayed. This study thus provides great insights into designing novel two-photon fluorescent materials with dual functionality and offers tools to understand the ER-stress related mechanism for cell and chemical biologists.