The invention belongs to the technical field of biomedicines and in particular relates to protein nanoparticles carrying an antitumor peptide and a preparation method and application of the protein nanoparticles. The preparation method comprises the following steps: according to gene sequences of a pET25b(+)-HSP16.5 expression vector, synthesizing a single stranded DNA (deoxyribonucleic acid) sequence corresponding to an apoptosis-promoting peptide, namely KLAK peptide, performing annealing treatment, performing HindIII and XhoI double-digestion on the product and the vector, performing gel recycling on the digestion product, connecting the KLAK sequence with the vector, transforming the connection product into a competence of DH5a escherichia coli, picking a monoclonal bacterial colony, performing overnight bacterium shaking, extracting a plasmid, and performing sequencing; and transforming a pET25b(+)-HSP-KLAK plasmid with correct sequencing into a competence of BL21 escherichia coli, amplifying culture, performing ultrasonic bacterium crushing so as to obtain a corresponding crude product, performing purification, and performing dialysis, concentration and freeze-drying, so as to obtain an HSP-KLAK protein. The heat shock protein nanoparticles carrying the antitumor peptide, which are provided by the invention, can be rapidly taken in by tumor cells, after being released inlysosome, a targeting mitochondrion plays a role of tumor killing, the protein of the medicine carrier self can be rapidly degraded by cells, and thus a good tumor treatment effect can be achieved.