The present work demonstrates how the fungal crude protein extract (FE) mediated gold nanoparticles (AuNPs) functionalized with aptamer (APT) improved cytotoxicity in human lung cancer cell line (A549) and brain tumor cell line (LN229). The agarose gel electrophoresis (AGE) confirmed the conjugation of APT in FE-AuNPs. The FTIR results revealed the involvement of FE in the amalgamation of FE-AuNPs. FE-AuNPs (lambda(max) 521 nm) had Z-average size of 19.72 +/- 0.26 d. nm and PDI of 0.58 +/- 0.01 with a zeta potential of -22.20 mV, whereas APT-FE-AuNPs (lambda(max)521 nm) had the Z-average size of 167.63 +/- 1.40 d. nm and PDI of 0.21 +/- 0.01 with a zeta potential of-36.36 mV. Both synthesized nanoparticles exhibited less cytotoxicity in normal NIH3T3 cells, while the APT-FE-AuNPs (lambda(max) 521 nm) showed significant cytotoxicity in A549 and LN229 cells. The cells treated with APT-FE-AuNPs (lambda(max) 521 nm) displayed higher percentage of early apoptosis (6.97 %), apoptosis (7.08 %), and necrosis (6.63 %) than those treated with bare or APT-FE-AuNPs (lambda(max) 565 nm) in A549 cells. The results demonstrated that the DNA aptamer (AS1411) tagged with FE-AuNPs (lambda(max) 521 nm) increased their accumulation in cancer cells, which triggered nucleolin targeted cell cytotoxicity through nucleus damage and induction of oxidative stress.