Atomic force microscopy is used to analyze the immobilization of single-stranded DNA (ssDNA) on poly-L-lysine (PL)-coated glass and subsequent hybridization with complementary DNA (dsDNA). 2D fractal analysis (power spectrum density algorithm (PSDA)) and line analysis methods are used to characterize the vertical aggregation of ssDNA/dsDNA. The nanodefects on the PL layer could be responsible for the vertical aggregation of DNA. The line analysis suggests that approximately two layers of ssDNA are adsorbed, stacked vertically. The PSDA is useful in understanding the direction of aggregation in addition to understanding the growth mechanism. Fractal dimension values are between 2 and 3, suggesting that the growth mechanism may obey the diffusion-limited aggregation (DLA) model.