Cotton fibers develop from epidermal cells in the outer integument of ovules. The regulatory process underlying fiber cell development has been extensively studied, little is known about the spatio-temporal profiles of transcriptomes and metabolomes during its development. Here we characterized the dynamics of transcriptome and metabolome during the early developmental stages of cotton fiber cells using a combination of spatial transcriptomic, single-cell transcriptomic, and spatial metabolomic analyses. We identified the key genetic regulators and metabolites that initiate and determine the fate of fiber cells. Both knockdown and gain-of-function analyses of one new identified marker gene BEE3/Gh_A09G062900 revealed its important role in the initiation of cotton fiber. We also designed a website for the public visualization of spatial gene expression in cotton that provides important reference datasets of spatio-temporal gene expression and can be used to further decipher the genetic and metabolic regulation of cotton fiber development (http://CottonOvule.cricaas.com.cn/).