In this work, polyaniline nanospindles have been synthesized using iron oxide as sacrificial template. These nanospindles were utilized for the fabrication of PANI-MoS2 nanoflower architectures via hydrothermal route. The electrostatic interaction between PANI and MoS2 improves the conductivity and provides more direct paths for charge transportation. SEM, TEM, XRD, Raman Spectroscopy techniques were employed to explore the crystal structure, and morphological properties of the PANI-MoS2 nanocomposite. Furthermore, an electrochemical biosensing platform based on PANI-MoS2 nanocomposite was fabricated for the specific detection of chronic myelogenous leukemia (CML) by using electrochemical impedance spectroscopy technique. The binding interactions between the pDNA/PANI-MoS2/ITO bioelectrode and target DNA sequence were also studied. The biosensor exhibits high sensitivity and wide detection range (10−6 M to 10−17 M) of target DNA with low detection limit (3 × 10−18 M). Additionally, the specificity studies of the genosensor with various target DNA sequences (complementary, noncomplementary and one base mismatch) and real samples analysis of CML shows its potential for clinical diagnostics.