In this study, a methodology to produce the high-quality gold nanoparticles (AuNPs) from aqueous Chinese lettuce (CL) leave extract as a reducing agent is presented. The effect of various parameters including Chinese lettuce (CL) leave extract, time, pH, temperature, and concentration of HAuCl₄ in AuNPs synthesis was studied using the UV–vis spectroscopy. The synthesized AuNPs were recognized with XRD, FTIR spectroscopy and also TEM analysis. In addition, antibacterial activity, antifungal activity, DPPH radical scavenging, ABTS radical scavenging, MTT assay, DAPI staining and flow cytometry against the A549 cells were examined. AuNPs synthesis produced a color change in the colloidal solution from light yellow to wine, and spectrum absorption showed the maximum peak about 521 nm indicating AuNPs formation. The optimum condition for AuNPs synthesis was 2 mM HAuCl₄.3H₂O and 3 ml CL leave extract at pH 6. The particle sizes of AuNPs were about 25 nm due to TEM results. XRD structural analysis confirmed that the nanoparticles have a crystalline structure with miller indexes (1 1 1), (2 0 0), (2 2 0) and (3 1 1) in the cubic network. Due to the FTIR analysis, the high stability of AuNPs can be attributed to the existence of CL leave extract on particles’ surfaces. No significant differences were observed in the cell viability at AuNPs concentration ranging from 0-2 μg/ml; however, A549 cells proliferation was strongly suppressed in a dose-dependent manner at concentrations higher than 4 μg/ml. Cytotoxicity results indicated that the viability of cells was 90% after 48 hours of treatment with AuNPs. Also, a healthy nucleus with less chromatin condensation after 48 h of treatment was observed. These findings suggested that AuNPs did not have adverse the effects on chromatin and DNA of A549 cells. The results of apoptosis assay indicated that AuNPs did induce necrosis and apoptosis in A549 cells. Although CL leave extract and AuNPs due to CL leave extract showed little antimicrobial activity, they indicated profound antioxidant activity against DPPH. The scavenging activity for DPPH and ABTS in AuNPs were more than CL leaves extract. This study indicated the promising potential of synthetized small sized and highly uniform AuNPs.