The monitoring of residual antibiotics in the environment has gained a significant importance for the effective control, because of the high risk to human health. A simple strategy was designed for the green synthesis and detection of doxycycline (Dox) by using anionic surfactant sodium bis 2-ethylhexylsulfosuccinate based silver nanoparticles (AOT-AgNPs). The chemical reduction and capping of Ag ions was achieved by sulfonyl and carbonyl functional groups of AOT molecule. The AOT-AgNPs were found to have excellent stability at variable environmental parameters (i.e. temperature, storage period, salt concentration and pH) possibly due to the strong emulsifying nature of the surfactant. Mechanism of interaction between the AOT-AgNPs and Dox was established with UV/visible, Fourier transform infrared (FTIR) spectroscopy, Atomic force microscopy (AFM) and Dynamic light scattering (DLS) techniques, which suggests the interaction via aggregates formation. The synthesize probe could detect the Dox within 15 min over a wide range of concentrations from 0.1 to 140μM with limit of detection (LOD) of 0.2 μM. As proof of strategy, we have illustrated that the AOT-AgNPs also detect Dox in biological and environmental samples with negligible interference and very significant recovery rates. Moreover, non-toxic nature against various tested cell lines (i.e. normal mouse fibroblast (NIH-3 T3) and cancerous non-small lung carcinoma (NCI-H460)) and significant antimicrobial, antibiofilm and biofilm eradicating potential of AOT-AgNPs were provide ideal nanomaterial for further applications.