The bombarding energy dependence of bonding structure in amorphous carbon interlayer and its effect on diamond nucleation density (Nd) were studied. Amorphous carbon (a-C) interlayer was deposited by magnetron sputtering. Interestingly, the intensity ratio (ID/IG) of the D band (∼1400 cm−1) to the G band (∼1570 cm−1) in the Raman spectra and the optical band gap of the a-C film were found to be inversely proportional to the sputtering power, that is, to bombarding energy. When diamond was subsequently deposited at 800 °C by microwave plasma chemical vapor deposition (CVD), diamond could be grown only on the interlayers with higher ID/IG (≥2.20), and Nd was increased up to 2 × 106/cm2 with the increase of ID/IG ratio, that is, with the decrease of the bombarding energy. We experimentally confirmed that the amount of the sp3 bonded carbon clusters within the interlayer was dependent on the bombarding energy of the particles, determining the diamond nucleation density. We suggest that the transformation of the amorphous carbon into graphitic carbon should be effectively prevented for the diamond nucleation on the a-C interlayer.