In enzymology, juglone 3-monooxygenase (EC 1.14.99.27) is an enzyme that catalyzes the chemical reaction

5-hydroxy-1,4-naphthoquinone + AH₂ + O₂ ${\displaystyle \rightleftharpoons }$ 3,5-dihydroxy-1,4-naphthoquinone + A + H₂O

The 3 substrates of this enzyme are 5-hydroxy-1,4-naphthoquinone, AH₂, and O₂, whereas its 3 products are 3,5-dihydroxy-1,4-naphthoquinone, A, and H₂O.

This enzyme belongs to the family of oxidoreductases, specifically those acting on paired donors, with O₂ as oxidant and incorporation or reduction of oxygen. The oxygen incorporated need not be derived from O miscellaneous. The systematic name of this enzyme class is 5-hydroxy-1,4-naphthoquinone,hydrogen-donor:oxygen oxidoreductase (3-hydroxylating). Other names in common use include juglone hydroxylase, naphthoquinone hydroxylase, and naphthoquinone-hydroxylase.

• Rettenmaier H, Lingens F (1985). "Purification and some properties of two isofunctional juglone hydroxylases from Pseudomonas putida J1". Biol. Chem. Hoppe-Seyler. 366 (7): 637–46. doi:10.1515/bchm3.1985.366.2.637. PMID 4041238.