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Flavonols are important copigments that affect flower petal coloration. Flavonol synthase (FLS) catalyzes the conversion of dihydroflavonols to flavonols. In this study, we identified a FLS gene, <i>MaFLS</i>, expressed in petals of the ornamental monocot <i>Muscari aucheri</i> (grape hyacinth) and analyzed its spatial and temporal expression patterns. qRT-PCR analysis showed that <i>MaFLS</i> was predominantly expressed in the early stages of flower development. We next analyzed the in planta functions of <i>MaFLS.</i> Heterologous expression of <i>MaFLS</i> in <i>Nicotiana tabacum</i> (tobacco) resulted in a reduction in pigmentation in the petals, substantially inhibiting the expression of endogenous tobacco genes involved in anthocyanin biosynthesis (i.e., <i>NtDFR</i>, <i>NtANS</i>, and <i>NtAN2</i>) and upregulating the expression of <i>NtFLS</i>. The total anthocyanin content in the petals of the transformed tobacco plants was dramatically reduced, whereas the total flavonol content was increased. Our study suggests that <i>MaFLS</i> plays a key role in flavonol biosynthesis and flower coloration in grape hyacinth. Moreover, <i>MaFLS</i> may represent a new potential gene for molecular breeding of flower color modification and provide a basis for analyzing the effects of copigmentation on flower coloration in grape hyacinth.