Rhamnolipid, among the most effective biosurfactants, is a glycolipid‐type biosurfactant primarily produced by Pseudomonas aeruginosa. In this study, rhamnolipid production was carried out using a strain of P. aeruginosa and it is aimed to compare rhamnolipid biopolymers obtained from various extraction methods using glycine (RG), hydrochloric acid (RH), diethyl ether (RD), ethyl acetate (RE). Comparison analyses were performed through NMR, FTIR techniques and viscosity, density measurements apart from determination of rhamnolipid yields. It can be concluded that rhamnolipid from diethyl ether for extraction is far from molecular structure to reference rhamnolipid molecule according to instrumental analyses performed. Besides, the yield of this rhamnolipid is much more than other rhamnolipids extracted through other methods but this is misleading because the value in there may be total sugar content apart from rhamnolipid. Therefore, RD extraction method can be said to be non‐selective process for rhamnolipid obtained. In RH method, some functional group peaks belonging to rhamnolipid were not observed. NMR analysis showed that some CH groups were not observed in the RG method. However, especially NMR and FTIR analyses showed that rhamnolipid obtained from RE method represented more accurate rhamnolipid based on reference molecule.Practical applications: This study showed that rhamnolipid production and its comparative analyses using various solvent extraction methods. Comparison analyses were carried out through NMR, FTIR techniques and viscosity, density measurements as well as determination of rhamnolipid yields. Different solvents affect seriously characteristics of rhamnolipid, which were defined in the previous literature reports. Especially, some methods are not selective ways for mentioning true biomolecule. To avoid misleading characterization information in the literature, these extraction methods were discussed through comparison methods such as NMR, FTIR analyses, and quantification measurements.Rhamnolipid biopolymers obtained from various extraction methods using glycine (RG), hydrochloric acid (RH), diethyl ether (RD), ethyl acetate (RE) are compared through NMR, FTIR techniques, and viscosity, density measurements. These analyses show that rhamnolipid obtained from RE method represents more accurate rhamnolipid based on reference molecule.