The ability to focus light on a subwavelength scale is essential in modern photonics. Optical microfiber-based sub-wavelength focusing will allow a miniaturized, flexible and versatile tool for many applications such as biomedical imaging and optomechanics. For a separate mode exited from an optical micro-/nanofiber endface, the photons will experience significant diffraction into the free space. This situation can be changed by incorporating two-mode interference along with the specific spatial distributions of both E-field amplitude and phase. Herein we report a novel approach to realizing sub-wavelength focusing based on the two-mode interference exited from an optical microfiber endface. By utilizing specific distributions of E-field amplitude and phase of two interacting optical modes, interference field patterns with a single focus (e.g., via a two-mode set of HE ₁₁and HE ₁₂) or multiple foci (e.g., via a two-mode set of HE ₁₁and HE ₃₁) can be obtained. Then, it is proved that the constructed foci will readily facilitate and selective trapping of nanoparticles. Circular polarization of optical mode is utilized in order to bring in angular symmetry of sub-wavelength focusing patterns compared with linear polarized optical modes. Our simulation results show that the smallest focal spot produced from the EH ₁₁and HE ₁₂mode interference has a full width at half-maximum (FWHM) of ~ 348 nm (i.e. 0.65 λ). Such a subwavelength focusing field is applied to the optical trapping of an 85 nm-diameter polystyrene nanosphere. Further calculation reveals that the stable trapping can be fulfilled with axial and transverse trap stiffness of 11.48 pN/(μm·W) and 64.98 pN/(μm·W), as well as axial and transverse potential well of 101 k _BT/W and 641 k _BT/W via two-mode interference of HE ₁₁and HE ₁₂. These values demonstrate the great improvement over conventional tapered fibers. Further investigations show that different foci, via a two-mode set of HE ₁₁and HE ₃₁, exhibit unlike trap stiffness and potential wells, justifying the potential for nanoparticle size sorting. Based on the flexible all-fiber device, this subwavelength focusing strategy by two-mode interference may find promising applications in optical manipulation, superresolution optical imaging, data storage and nanolithography.