Optical tweezers have broad applications in studies of structures and processes in molecular and cellular biophysics. Use of optical tweezers for quantitative molecular-scale measurement requires careful calibration in physical units. Here we show that DNA molecules may be used as metrology standards for force and length measurements. Analysis of DNA molecules of two specific lengths allows simultaneous determination of all essential measurement parameters. We validate this biological-calibration method experimentally and with simulated data, and show that precisions in determining length scale factor ( approximately 0.2%), length offset ( approximately 0.03%), force scale factor ( approximately 2%), and compliance of the traps ( approximately 3%) are limited only by current measurement variation, much of which arises from polydispersity of the microspheres ( approximately 2%). We find this procedure to be simpler and more convenient than previous methods, and suggest that it provides an easily replicated standard that can insure uniformity of measurements made in different laboratories.