It is very important to develop a rapid, simple, low cost point-of-care (POC) method for the early diagnosis of pathogens. In this work, a flexible paper-based electrode based on nickel metal-organic framework (Ni-MOF) composite/Au nanoparticles/carbon nanotubes/polyvinyl alcohol (Ni-Au composite/CNT/PVA) was constructed to detect target human immunodeficiency virus (HIV) DNA by DNA hybridization using methylene blue (MB) as a redox indicator. The CNT/PVA and Ni-Au composite were deposited on the cellulose membrane by vacuum filtration and drop-coating method in turn to obtain Ni-Au composite/CNT/PVA (CCP) film electrode. Compared to the CNT/PVA film electrode, CCP film electrode makes a higher loading of the probe DNA for its large specific surface area and conjugated π-electron system that can provide hydrogen bond sources to achieve interactions between MOF and single-stranded DNA, which improves the sensitivity for detecting target DNA. The variation of peak current for MB molecules adsorbed onto DNA before and after hybridization with HIV DNA was monitored. Electrochemical results proved that the CCP film maintained stable electrochemical property even after bending 200 times or stretching under different strains from 0% to 20%. The flexible paper electrode showed excellent sensing performance with a linear range of 10 nM-1 μM and a low detection limit of 0.13 nM. The target HIV DNA was successfully detected even in complex serum samples using the flexible CCP film electrode. Therefore, the simple and inexpensive flexible paper-based MOF composite film electrode can also be utilized for other pathogens POC diagnosis.