Thermally activated delayed fluorescence (TADF) materials have garnered significant attention as potential candidates for biological photoluminescence imaging. However, the achievement of tunable chiral TADF materials through aggregated and self-assembled processes remains a formidable challenge. In this study, four donor-acceptor-donor type chiral TADF molecules, designed by directly linking electron-donating moieties (phenoxazine, phenothiazine or 1-phenyl-1,2,3,4-tetrahydroisoquinoline) with an electron-acceptor unit (diphenyl sulfone), as promising candidates for luminescent imaging applications are presented. The experimental investigation reveals that these chiral TADF materials possess remarkably small Delta EST values, promoting efficient reverse intersystem crossing (RISC). Additionally, they exhibit aggregation-induced emission and long-delayed luminescent lifetimes affected by the aggregated and self-assembled state. Moreover, deoxygenation significantly enhances the emission, enabling high-contrast hypoxia probing, which shows great potential for high-contrast photoluminescence imaging in living cells. This work not only offers a molecular design strategy for chiral TADF materials but also extends the diverse biological applications of high-contrast hypoxia detection in the biological field.