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Due to the scarcity of C–F bond-forming enzymatic reactions in nature and the contrasting prevalence of organofluorine moieties in bioactive compounds, developing biocatalytic fluorination reactions represents a pre-eminent challenge in enzymology, biocatalysis and synthetic biology. Additionally, catalytic enantioselective C(sp3)–H fluorination remains a challenging problem facing synthetic chemists. Although many non-haem iron halogenases have been discovered to promote C(sp3)–H halogenation reactions, efforts to convert these iron halogenases to fluorinases have remained unsuccessful. Here we report the development of an enantioselective C(sp3)–H fluorination reaction, catalysed by a plant-derived non-haem enzyme 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO), which is repurposed for radical rebound fluorination. Directed evolution afforded a C(sp3)–H fluorinating enzyme ACCOCHF displaying 200-fold higher activity, substantially improved chemoselectivity and excellent enantioselectivity, converting a range of substrates into enantioenriched organofluorine products. Notably, almost all the beneficial mutations were found to be distal to the iron centre, underscoring the importance of substrate tunnel engineering in non-haem iron biocatalysis. Computational studies reveal that the radical rebound step with the Fe(III)–F intermediate has a low activation barrier of 3.4 kcal mol−1 and is kinetically facile.