Optical tweezers have increasingly widespread application in a large variety of scientific disciplines because they can trap a micro-particle losslessly. They can also trap multiple particles in single optical trap due to the characteristic of the potential energy of the trap. How to distinguish the number of particles in single optical trap is the key work in the experiment. A novel method based on laser scattering method to distinguish the numbers of nanometer-particles in single trap was presented in this paper. The number of particles in single trap was distinguished by measuring the intensity of light backscattering with corresponding situation. The number of particles with diameters of 1 μm,0.5 μm,0.2 μm,100 nm,73 nm was distinguished successfully,and the state of optical trap was discussed. This method may find the application of optical tweezers in nanometer dimension.