Use of biosurfactant as a green stabilizer for the biological synthesis of gold nanoparticles (AuNPs) is now emerging as a nontoxic and environmentally acceptable "green chemistry" procedure. Stability of AuNPs at different pHs is very important because our body has different pHs. This paper addresses this issue. In this work, first P. aeruginosa PTCC 13401 was used to produce rhamnolipid biosurfactant. The highest rhamnolipid production occurred at 120 h, achieving a value of 3.1 g/L. The thin layer chromatography (TLC) indicated that the crude product is a mixture of mono-rhamnolipid and di-rhamnolipid with retardation factor (Rf) value of about 0.35 and 0.78, respectively. Moreover, rhamnolipid solutions with different pHs were added to HAuCl4 solution and incubated for 24 h at 37 oC and 150 rpm. The formation of spherical AuNPs was monitored using a UV–vis spectrophotometer and verified by TEM. Our results showed that the formation of AuNPs occurred just for pH values between 7.0 -8.0. Measurement of the surface tension of the solution at different pH values was performed to find out the reason for this observation. Our results showed that the surface tension was also stable only between pH 7.0-8.0. This was inferred from precipitation of rhamnolipid at higher and lower pH values. The results of this work may help pharmacists to have a good prediction of behavior of rhamnolipid biosurfactants as a green stabilizer for biomedical applications including tissue engineering and drug delivery.