The mechanism of electron-beam-induced immobilization of nanoparticles on a substrate has been studied both experimentally and theoretically. Experiments have been performed for the case of 200–350 nm Co–Ni nanoparticles secured to a substrate using a 30 keV electron beam. Atomic force microscopy studies reveal that the fixing occurs due to the formation of a deposit beneath the nanoparticles, causing strong bonding to the substrate, even for a thin layer. Measurements of the lateral forces required to displace the immobilized nanoparticles have shown that a deposit layer of 0.5 nm results in a tenfold increase in the bonding strength. A comparison of measured profiles with the results of computer simulations clearly reveals that the major role in the formation of the deposit is played by low-energy electrons generated by energetic primary electrons in both the nanoparticles and substrate. It is also shown that the efficiency of bonding decreases with decreasing energy of primary electrons. Different strategies for electron-beam-induced immobilization of nanoparticles and optimization of the processes are discussed.