The invention discloses a preparation method of a sample with standard fiber component content. The preparation method comprises the following steps: 1) fiber shortening, 2) drying, 3) weighing, 4) evenly mixing and stirring, 5) filtering, 6) shaping, 7) pressing, 8) drying, 9) assigning a dried sample, labeling a name and a proportion of content of fiber, so that the fiber standard sample is obtained. According to the preparation method of the sample with the standard fiber component content, a sample of various fiber combinations can be prepared flexibly in a mass mode, cost of preparation of the sample is low, and operation method is simple. The prepared sample is accurate in fiber content, good in uniformity and shaping, and capable of being used as a standard sample for creating a model base fixed mold when the fiber component is measured by creating near infrared spectroscopy analysis, being used in a lab and used as a sample for detection quality control on the fiber component through a chemical dissolution method, being used as a standard sample for quality control on fiber samples such as cotton-hemp blended products and blended products by cashmere and woolens or special animal hairs, wherein the fiber samples cannot be quantified through the chemical dissolution method.