Chiral assemblies are increasingly recognized for their ability to dictate photophysical processes, notably by enhancing triplet generation in intercalated chromophores for applications in phototherapeutics and chiral optoelectronics. However, the precise mechanism of chirality-enhanced intersystem crossing (ISC) remains elusive, as deciphering it requires resolving the complex coupling between ultrafast excited-state dynamics and the evolution of excited-state chirality. Herein, by integrating femtosecond time-resolved circularly polarized luminescence (fs-TRCPL) spectroscopy with transient absorption (TA) spectroscopy and quantum chemical calculations, we directly correlate the chiral gradient imposed by DNA assemblies with enhanced ISC kinetics for the first time. Using a natural antibiotic gilvocarcin V (GV) intercalated into a B-typed dA18•dT18 duplex, a pronounced increase of triplet quantum yield of GV from 7.6% in the solution to 25.8% in DNA is observed. Such enhancement is driven by an amplification of GV's excited-state chirality, leading to direct ISC from the Franck-Condon (FC) region and spin-orbit charge transfer ISC from charge-transfer state simultaneously. This work elucidates the mechanism of excited-state chirality on triplet-state generation and offers a novel design principle for high-performance triplet sensitizers leveraging chiral assemblies.