Abstract: We have been developing a magnetic immunoassay method using magnetic markers and magneto-resistive (MR) sensor for liquid-phase detection of biological targets. In this method, bound and unbound (free) markers can be magnetically differentiated by using the difference in their Brownian relaxation times. In order to improve the detection sensitivity, we developed two methods. First, we clarified how the signals from the bound and free markers are affected by the concentration of the magnetic markers. We found the most suitable concentration was 25 μg/mL. Next, we developed dispersion process after applying the excitation field. With this dispersion process, we can dissolve agglomerate of the free markers caused by the excitation field. As a result, blank signal from the free markers can be much decreased. Using the developed methods, we performed the liquid-phase detection of biotins. The minimum detection number of biotins was as low as N_<B,min> = 1.3×10^5 existing in 60 μL sample solution. The molecular number concentration was 3.6×10^<-18> mol/ml, indicating high sensitivity of the present method.