Cotton is the most important textile fiber crop in the world. With expect to improve cotton fiber yield, many studies have tried to increase fiber number per seed. Estimating mature fiber number exactly is becoming more necessary, because only about thirty percentages of differentiated fiber cells could develop to mature fibers. To date, there is no method for counting mature fibers, except some ones for early fiber cells. Here, we describe a fast and easy approach for counting mature fibers per seed. That would help us finding the change of fiber number during cotton improvement.