Drug safety for human body should be carefully studied for its potent clinical application. In this report, the neurotoxicity of anticancer drug daunorubicin (DNR) and the oleic acid-capped Fe3O4 nanoparticles (NPs) for rat brain was firstly explored by using the in vivo microdialysis. The results indicated that the anticancer drug DNR itself had the serious neurotoxicity for the rat brain. And this neurotoxicity was influenced through the concentration changes of amino acids. The concentration level of some excitatory amino acids (such as Glu) and some inhibiting amino acid (such as Gly) were considerably decreased while that of the excitatory amino acid Asp was remarkably increased. For the DNR conjugated with Fe3O4 NPs nanocomposites, the side effect of DNR was visibly cut down, and the time to cause the side neurotoxicity was apparently shortened. Thus, it is evident that compared with DNR alone, the DNR conjugated with Fe3O4 NPs nanocomposites have the better biocompatibility and bio-security for the relevant cancer treatment in vitro and in vivo. This raises the promising possibility of the application of these DNR conjugated with Fe3O4 NPs nanocomposites for the target cancer therapy.