Background:Application of nanoenzymes, based on D-amino acid oxidase (DAAO) conjugated to magnetic nanoparticles (NPs), as anticancer system requires improvement of the synthesis protocol and in vivo distribution evaluation.Results:A new and more efficient synthesis via EDC-NHS produced an Fe3O4NP-APTES-DAAO system with a specific activity of 7 U/mg NPs. IR spectroscopy showed that all Fe3O4 NP sites are saturated with APTES and all available NH2 sites with DAAO. The acute cytotoxicity of the new system does not differ from that of the previous one. In vivo experiments showed that the system did not cause adverse effects, cross the brain–blood barrier and accumulate in the heart.Conclusions:Our results support the possibility to use enzymes conjugated to magnetic NPs for cancer treatment. Besides, we think that enzymes and other biological molecules efficiently conjugated to magnetic NPs might constitute a category of ‘bionanoparticles’ to be exploited, not only in medical, but also in industrial biotechnology.