One of the main problems faced by engineers in EOR is not having the conditions for the formation of Winsor III (ultralow IFT), which causes a reduction in oil recovery efficiency and also an increase in costs. Therefore, this research tries to fill this gap through the smart injection of biosurfactants into oil reservoirs. To simulate Winsor III, the aqueous phase [NaCl (10,000−150,000 ppm) and rhamnolipid (300 mg/L)] and crude oil (API°31) were mixed in a 1:1 ratio (v/v) in glass test tubes. In this work, rhamnolipid biosurfactant was first produced by Pseudomonas aeruginosa PTCC 1340 and afterward confirmed using FTIR, NMR, and TLC analyses. The produced rhamnolipid reduced IFT from 28.4 (oil phase/brine) to 1.8 mN/m, and the slab's wettability changed from strongly oil-wet (θ = 146.2°) to intermediate-wet (θ = 92.4°). Our results showed that ultralow IFT (Winsor III) was obtained at about 0.015 mN/m under conditions of salinity of 40,000 ppm, rhamnolipid concentration of 300 mg/L, and ambient temperature. Core flooding tests in carbonate cores led to substantial tertiary recovery of 18.1 and 14.8% of OOIP (Original Oil in Place) under Winsor III and II, respectively, over conventional water flooding. This increase in EOR can be due to the increase in the capillary number. According to the proposed novel method in this research, the capillary number of Winsor III increased about 8 times compared to that of Winsor II through the smart injection of biosurfactant into the core. Our results are an initial step to help researchers through smart injection in oil reservoirs to provide the possibility of being in Winsor III conditions to reach the ultralow IFT and subsequently increase oil recovery efficiency.