The invention belongs to the technical field of medicines, and in particular relates to a nanosuspension of annonaceous acetogenin drugs prepared from such amphiphilic stabilizers as mPEG PCL, mPEG PLA, mPEG PLGA, mPEG DSPE, mPEG Chol, SPC, Tween80, BSA, TPGS and the like, as well as a preparation method and an application of the nanosuspension. The annonaceous acetogenin total lactone nanosuspension is prepared by virtue of a solvent precipitation-ultrasonic injection method, and according to a prescription, the proportioning ratio of the annonaceous acetogenin drugs to the stabilizer is at 1 to (0.02-10) (in percentage by weight). The prepared annonaceous acetogenin nanosuspension can reach a load-loading capacity to 90% to the greatest extent and can reach a minimum grain size to 123.2nm, and the nanosuspension is good in polydispersity. The nanosuspension is stable in both gastrointestinal fluid and plasma, and the nanosuspension is applicable to oral administration and injection administration; the nanosuspension has a good in-vitro sustained-release effect and is free from burst release; the nanoparticle, in comparison with a crude drug solution, is more obvious in tumor cell inhibitory rate in vitro; the nanosuspension, in in-vivo tissue distribution, shows tumor passive targeting; therefore, the nanosuspension is beneficial for enhancing efficacy and reducing toxic and side effects. Meanwhile, an in-vivo efficacy experiment shows that the nanosuspension is outstanding in antineoplastic efficacy, and the nanosuspension has a broad development prospect.