In this study, we examined the dependence of fluorescent intensity on different factors in the synthesis process of Fe3O4-cyanine 5.5 nanoparticles. The results showed that Fe3O4 nanoparticles needed to be covered with a layer of NH2 group before reacting with cyanine 5.5-NHS ester. The best cyanine 5.5 NHS ester:Fe3O4 and cyanine 5.5 NHS ester :APTES ratios were 1:20 and 1:3, respectively. The drug delivery did not affect much on the fluorescent property while increasing in excitation wavelength could lead to increase in fluorescent intensity of the system.