A nitrocellulose–graphene oxide hybrid that consists of a commercially nitrocellulose (NC) membrane non-covalently modified with graphene oxide (GO) microparticles was successfully prepared for oligonucleotide extraction. The modification of NC membrane was confirmed by Fourier Transform Infrared Spectroscopy (FTIR), which highlighted the principal absorption bands of both the NC membrane at 1641, 1276, and 835 cm−1(NO2) and of GO in the range of 3450 cm−1(CH2-OH). The SEM analysis underlined the well-dispersed and uniform coverage of NC membrane with GO, which displayed thin spider web morphology. The wettability assay indicated that the NC–GO hybrid membrane exhibited slightly lower hydrophilic behavior, with a water contact angle of 26.7°, compared to the 15° contact angle of the NC control membrane. The NC–GO hybrid membranes were used to separate oligonucleotides that had fewer than 50 nucleotides (nt) from complex solutions. The features of the NC–GO hybrid membranes were tested for extraction periods of 30, 45, and 60 min in three different complex solutions, i.e., an aqueous medium, an α-Minimum Essential Medium (αMEM), and an αMEM supplemented with fetal bovine serum (FBS). The oligonucleotides were desorbed from the surface of the NC–GO hybrid membrane using Tris-HCl buffer with a pH of 8.0. Out of the three media utilized, the best results were achieved after 60 min incubation of the NC–GO membranes in αMEM, as evidenced by the highest fluorescence emission of 294 relative fluorescence units (r.f.u.). This value corresponded to the extraction of approximately 330–370 pg (≈7%) of the total oligo-DNA. This method is an efficient and effortless way to purify short oligonucleotides from complex solutions.