The change in position of fluorescent beads captured inside the focal volume of optical tweezers is monitored using fluorescence emission induced by two‐photon absorption of a continuous‐wave Nd:YAG laser (λ=1064 nm). The displacement of a bead due to interactions with its environment leads to a fluorescence intensity variation that is used to design a novel spatial sensor. We determine changes in the axial position of a CY3‐labeled latex bead with a diameter of 1.03 μm to a precision better than 10 nm. At an intensity of 600 mW/ μm2 the two‐photon bleaching rate is lower than 50% per 2000 s.