The solvent polarity effect on chain conformation, film morphology, and photophysical properties of a nonionic water-soluble conjugated polymer (WSCP), poly[2,5-bis(diethylaminetetraethylene glycol)phenylene vinylene] (DEATG-PPV) is investigated in detail. The combination of stationary absorption and photoluminescence (PL) spectroscopy, time-resolved PL spectroscopy, and fluorescence correlation spectroscopy methods enables us to probe the chain conformation of DEATG-PPV, down to the level of a single chain when working with extremely diluted solutions. The use of correlated atomic force microscopy and confocal fluorescence lifetime imaging microscopy measurements of drop-casted DEATG-PPV films reveals the intrinsic relationship between chain conformation, film morphology, and optical properties. Depending on solvent polarity, DEATG-PPV presents extended, coiled, and collapsed chain conformations in solutions, which lead to distinct morphology and optical properties in solid films. Our work presents a pathway to control and characterize the film morphologies of WSCPs toward the optimal performance of various optoelectronic devices.