Disclosed is an ossotide injection and preparation method, wherein the preparation comprises the following steps, (1) extracting from the bone of mammals through salt solution heat pressing and boiling, centrifugal filtrating, merging the extract, dispensing 1-40 hours at low-temperature, subjecting the raffinate to centrifugal filtration, collecting the filtrate for later use, (2) vacuum concentrating the filter liquor and charging ethanol, stewing and settling, filtering the upper layer alcohol liquid, reclaiming ethanol, obtaining water extract, (3) charging distilled water of the equal volume, passing in turn through weakly-acidic cationic resin column, weakly-basic anionic resin column, pearl form chitosan column and chitosan modified activated charcoal column, rouging and purifying, obtaining pure thin soup, (4) concentrating the pure thin soup, passing through microporous filtration film under pressurization, loading the filtrate, sealing and hot-pressed sterilizing, or freezing and drying the obtained soup, thus obtaining freeze-dried powder injection.