Herein, the synthesis of a biocompatible silver nanoparticles (AgNPs), for colorimetric detection of toxic mercury (II) ion (Hg2+), is reported. Phenolic-rich fraction of Asystasia gangetica leaf was extracted and used as a reductant of silver salt, all within the hydrophilic konjac glucomannan (KgM) solution as stabilizer, at room temperature (RT). The bioactive components of Asystasia gangetica phenolic extract (AGPE), as elucidated with a (UHPLC-MS-QTOF-MS), revealed plethora of phenolic compounds, which can facilitate the reduction of silver salt at ambient conditions. Sparkling yellow colloidal solution of KgM-AgNPs was realized within 1 h, at RT, having a UV–vis maximum at 420 nm. KgM-AgNPs was characterized using UV–vis, Raman and (FTIR), TEM, SEM, EDS, XRD, TGA/DTG. TEM and FESEM images showed that KgM-AgNPs were spherical, with particle size distribution around 10–15 nm from TEM. The KgM-AgNPs biocompatibility was investigated on mouse L929 fibrobroblast and rat erythrocytes, without any harmful damages on the tested cells. In aqueous environment, KgM-AgNPs demonstrated good detection capacity toward Hg2+, in a Hg2+ concentration dependent fashion, within 3 min. Absorbance ratios (A360/A408) was linear with Hg2+ concentrations from 0.010–10.0 to 10.0–60.0 µM, with an estimated (LOD) of 3.25 nM. The probe was applied in lake water sample, with satisfactory accuracy.