Laser-induced thermal effects in optically trapped microspheres and single cells are investigated by quantum dot luminescence thermometry. Thermal spectroscopy has revealed a non-localized temperature distribution around the trap that extends over tens of micrometers, in agreement with previous theoretical models besides identifying water absorption as the most important heating source. The experimental results of thermal loading at a variety of wavelengths reveal that an optimum trapping wavelength exists for biological applications close to 820 nm. This is corroborated by a simultaneous analysis of the spectral dependence of cellular heating and damage in human lymphocytes during optical trapping. This quantum dot luminescence thermometry demonstrates that optical trapping with 820 nm laser radiation produces minimum intracellular heating, well below the cytotoxic level (43 °C), thus, avoiding cell damage.