Copper-substituted nickel manganites Ni(1−x)CuxMn2O4 (Ni-TCE-NPs) were produced by co-precipitation route (sol–gel) at room temperature. Ni(1−x)CuxMn2O4-Bio (NCB) NPs were studied by powder X-ray diffraction technique, scanning electron microscopy and Raman spectroscopy. XRD spectra authenticated the copper-doped nickel manganites’ formation with particle size 23–28 nm. A significant decrease in the lattice parameter confirmed the doping of copper ions into the nickel manganites. Microscopy (SEM) was used to estimate the grain size, shape and uniformity, revealing the non-uniform agglomerated polygon and plate-like microstructure. The NCB-NPs showed anticoagulant activity by enhancing the coagulation time of citrated plasma of human beings. NCB-NPs with x = 0.35 and 0.45 have increased clotting time from control 133 ± 4 s to 401 ± 7 s and 3554 ± 80 s, respectively, and others around 134 s. Additionally NCB-NPs with x = 0.35, 0.45 inhibited the platelet aggregation by 80% and 92%, while remaining inhibited with only 30%. NCB-NPs did not show hemolytic activity in RBC cells intimate its non-toxic nature. Finally, NCB-NPs were non-toxic and known to exhibit anti-blood-clotting and antiplatelet activities, which can be used in the field of biomedical applications, especially as antithrombotic agents.