Cotton is one of the most important cash crops worldwide. Chlorophyll is a critical photosynthetic pigment in plants, which could significantly affect the growth and development of cotton. In this study, one insertion mutant, ZD131, was found to have darker leaves and higher chlorophyll content than wild-type (WT) at the seedling and flowering stages. The chlorophyll contents were also higher in F1 and F2 heterozygous plants crossed by ZD131 and WT than that in WT. Through genome resequencing of mutant ZD131, a coding gene Ghir_D07G020490, we named it GhFP, was found at 2347 bp downstream of the insertion site in ZD131, which has not been studied yet. GhFP was localized in the cytoplasm and expressed higher in ZD131 than that in WT. Furthermore, virus-induced gene silencing (VIGS) of GhFP in cotton and overexpression of GhFP in Arabidopsis verified that GhFP could positively regulate the chlorophyll content and biomass. Therefore, this study revealed that a novel gene GhFP could regulate chlorophyll biosynthesis and biomass accumulation, which laid a theoretical foundation for understanding the regulatory mechanism of chlorophyll accumulation and provided essential gene resources for cotton genetic improvement.