The invention belongs to the technical field of medicines, relates to a pharmaceutical composition of a naloxone hydrochloride injection and a preparation method of pharmaceutical composition, and particularly relates to a pharmaceutical composition of a naloxone hydrochloride injection. The pharmaceutical composition comprises naloxone hydrochloride, and particularly comprises naloxone hydrochloride, saccharides, organic acid and injection water. The naloxone hydrochloride in the pharmaceutical composition is added to the composition in a form of 17-allyl-4,5alpha-epoxy-3,14-dihydroxy-pyran-6-methadone hydrochloride dehydrate; the saccharides are selected from one or more of the following components: mannitol, sorbitol, lactose, maltose, glycine, mycose, glucose and the like. The pharmaceutical composition disclosed by the invention can be used after opioid compound anaesthesia to resist respiratory depression caused by the medicine and prompt patients to revive, is used for respiratory depression caused by excessive opioids and complete or partial reversion of the opioids, is used for saving acute alcohol poisoning, and is used for diagnosing excessive acute opioids.