Acinetobacter baumannii
is the main cause of nosocomial infections in blood, urinary tract, wounds and in lungs leading to pneumonia. Apart from its strong predilection to be the cause of serious illnesses in intensive care units. Herein, we present a specific and sensitive approach for the monitoring of
Acinetobacter baumannii
genome based on citrate capped silver nanoparticles (Cit-AgNPs) using spectroscopic methods. In this study, (5ʹ SH-TTG TGA ACT ATT TAC GTC AGC ATG C3ʹ) sequence was used as a probe DNA (pDNA) of
Acinetobacter baumannii
. Then, complementary DNA (cDNA) was used for hybridization. After the hybridization of pDNA with cDNA, target DNA (5ʹ GCA TGC TGA CGT AAA TAGTTC ACA A 3ʹ) was recognized and detected using turn-on fluorescence bioassay. After the hybridization of pDNA with cDNA, the target DNA was successfully measured in optimum time of 2 min by spectrophotometric techniques. Moreover, the selectivity of designed bioassay was evaluated in the presence of two mismatch sequences and excellent differentiation was obtained. 1 Zepto-molar (zM) of low limit of quantification (LLOQ) was achieved by this genosensor. The present study paved the way for quick (2 min) and accurate detection of
Acinetobacter baumannii
, which can be a good alternative to the traditional methods. Current study proposed a novel and significant diagnostic test towards
Acinetobacter baumannii
detection based on silver nanoparticles aggregation which has the capability of being a good alternative to the traditional methods. Moreover, the proposed genosensor successfully could be applied for the detection of other pathogens.