Diamond nucleation by biased hot filament chemical vapor deposition was investigated by scanning electron microscopy and atomic force microscopy. It was found that a number of microdefects were produced on a substrate surface owing to energetic ion bombardment under negative substrate bias, which increased with increasing negative bias. The nucleation density was enhanced with an increase of negative bias. During diamond nucleation, a purple glow was observed when the negative bias was increased to a critical value. At the onset of glow discharge, the process of diamond nucleation on a silicon surface by biased hot filament chemical vapor deposition was theoretically studied by analysis of the experimental results of diamond nucleation. The relationship among the number of active ions, the microdefects, and nucleation density with negative bias was given by reasonable analytic formulas. The effect of negative bias on ion diffusion on the substrate surface was theoretically researched and deduced. The influence of negative bias on the bond strength of diamond nuclei on the substrate was analyzed theoretically. The adhesion force between diamond nuclei and the substrate surface was measured by means of the scratch surface method, which was in accord with theoretical consideration. The results indicated that the theoretical calculation was in agreement with experimental results.