A determination method of the 1-deoxynojirimycin content in silkworm powder relates to silkworm powder. The method comprises the following steps: weighing two 1-deoxynojirimycin control samples used as control liquid 1 and 2; sucking the control liquid 1 and 2, injecting the samples; weighing silkworm powder to be determined, adding petroleum ether, performing heating reflux and extraction in water bath, filtering, removing the filtrate, volatilizing the petroleum ether to dryness to obtain a product A; adding methanol in A, performing ultrasonic extraction, filtering, concentrating the filtrate, adding in a neutral alumina column, performing elution, collecting the eluted solution, evaporate to dryness, dissolving by methanol and obtaining a constant volume so as to used the solution as a test solution; performing HPLC analysis of the test solution, calculating the 1-deoxynojirimycin content in the silkworm powder to be determined by an external standard two-point method according to a peak area value. The method has the advantages of high precision, good repeatability and stability, qualified sample recovery rate, simplicity and practicality, and the like, can be popularized in general laboratories, and is applicable to the quality control of 1-deoxynojirimycin in silkworm powder products.