Orthorhombic boron nitride film is prepared on Si(100) substrate by radio frequency plasma enhanced pulse (Nd:YAG) laser deposition (RF-PEPLD) in Ar-N2 gas system, assisted with substrate pulse negative bias -150v, substrate temperature of 500°C and deposition time of 30 minutes. The phase compositions of the film are characterized by Fourier transform infrared (FTIR) spectroscopy, glancing-angle X-ray diffraction (XRD), Raman spectroscopy and scanning electron microscopy (SEM). The results show that high quality orthorhombic boron nitride film has been prepared. A layer structure growth mechanism of orthorhombic boron nitride phase upon RF-PEPLD is discussed in this paper. A thin layer h-BN  is deposited before depositing o-BN and h-BN mixed phase, then o-BN percentage composition of the BN film becomes creasing.