The invention discloses a colored cotton ovule in vitro culture method, which comprises the steps of: collecting 0-2 days old young bolls of green cotton or brown cotton of the day, pulling off the corolla, stripping calyxes, and retaining flower stalks; washing the young bolls with distilled water twice, conducting disinfection with 75% alcohol for 5-15min under a sterile condition, then performing soaking with 0.1% mercury chloride for 5-15min, and conducting washing with sterile water 5-8 times, and stripping ovules; inoculating the ovules under a sterile condition into a BT medium containing hormone ZR, GA3 and IAA, inoculating 15-25 ovules of one ovary into each bottle, and carrying out dark culture for 30-40d at 30-32DEG C. Preferably, during inoculation of green cotton ovules, the hormone ratio wais GA31.0micromol / L+IAA5.0micromol / L+ZR2.5micromol / L, and during inoculation of brown cotton ovules, the hormone ratio is GA31.0micromol / L+IAA1.0micromol / L+ZR1.0micromol / L. The method provided by the invention can reduce the contamination rate, improve the survival rate, and promote cotton fiber growth and pigment synthesis.