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Many existing in vitro biosystems harness power from the chemical energy contained in substrates and co‐substrates, and light or electric energy provided from abiotic parts, leading to a compromise in atom economy, incompatibility between biological and abiotic parts, and most importantly, incapability to spatiotemporally co‐regenerate ATP and NADPH. In this study, we developed a light‐powered in vitro biosystem for poly(3‐hydroxybutyrate) (PHB) synthesis using natural thylakoid membranes (TMs) to regenerate ATP and NADPH for a five‐enzyme cascade. Through effective coupling of cofactor regeneration and mass conversion, 20 mM PHB was yielded from 50 mM sodium acetate with a molar conversion efficiency of carbon of 80.0 % and a light‐energy conversion efficiency of 3.04 %, which are much higher than the efficiencies of similar in vitro PHB synthesis biosystems. This suggests the promise of installing TMs as a green engine to drive more enzyme cascades. [ABSTRACT FROM AUTHOR]