To remove methylene blue (MB) from water, rhamnolipid-modified biochar (BC-RL) was synthesized via a facile method. The surface structures and properties of the biochar (BC) and BC-RL were characterized by scanning electron microscopy, Fourier transform infrared spectra, X-ray diffraction spectra, X-ray photoelectron spectra, N2 adsorption-desorption isotherms, and Raman spectra. The results showed that modification with rhamnolipid remarkably increased the functional groups on the BC-RL, but reduced the surface area. The MB removal efficiency of the BC-RL increased with an increase in the adsorbent loading and temperature. Moreover, the adsorption performance of the BC-RL was obviously higher than that of the BC, which was mainly attributed to the increased number of functional groups. The adsorption kinetic data was fitted well to the pseudo-second-order model with a coefficient of determination greater than 0.999. The coefﬁcients of determination of the adsorption isotherm fitted by different models decreased in the following order: BET > Freundlich > Langmuir. This indicated that the adsorption of MB onto the BC-RL involves a multilayer formation process. These results suggested that the BC-RL could be an environmentally benign and cost-effective adsorbent for the removal of MB from aqueous solutions.