We report the design and synthesis of a novel pyrido[1,2-<i>a</i>]indole fluorophore that exhibits deep-red emission (up to 660 nm), a large Stokes shift (6118 cm<sup>-1</sup>) in methanol, and excellent biocompatibility. Positional nitration enables systematic modulation of the photophysical properties across a series of regioisomers. Spectroscopic analyses and calculations revealed the intramolecular charge transfer (ICT) and rotation-induced quenching behavior. The fluorophores showed exceptional lipid droplet (LD) labeling capability in live-cell imaging without washing steps, indicating their potential for biomedical applications. This study demonstrates that simple nitro-group relocation on a compact heteroaromatic scaffold provide tunable, environment-sensitive fluorophores for probe development.