Novel methods for rapid, sensitive and low-cost biomolecule detection have attracted particular interest because of their wide use in medical diagnostics, food inspection and biomedical research applications. gold nanoparticle (aunps) colorimetric assay using aptamers is a low cost and a highly effective means for detecting a wide range of biomolecular targets. a simple and stable rna aptamer based colorimetric sensor for the detection of vitamin b12 using aunps was proposed. vitamin b12 belongs to the b vitamin group and prevents pernicious anemia, which is caused by vitamin b12 deficiency. a highly stable rna aptamer that bind to vitamin b12 was employed by structural modification at 2’-hydroxyl group of ribose to 2'-flouro of all pyrimidines indicated in lowercase in 35 bases length aptamer (5’ gga acc ggu gcg cau aac cac cuc agu gcg agc aa 3’). aggregation of aunps was specifically induced by desorption of the vitamin b12 binding rna aptamer from the surface of aunps as a result of the aptamer target interaction, leading to the color change from red to purple. the level of detection of vitamin b12 was 0.1 μg/ml by successful optimization on the amount of the aptamers, aunps, salts, and stability of aptamer. analysis of vitamin b12 was carried out, and the observed recovery was 92–95.3% with a relative standard deviation in the range of 2.08–8.27 %.the results obtained were compared with those of the uv-vis spectrometry method. this colorimetric aptasensor is advantageous for onsite detection with naked eye.