Using nano imprinting technology, a scale tool was fabricated for evaluating image properties of a biological fluorescence microscope. Evaluation of the properties given by a laser scanning microscope could be demonstrated. On this scale, a minute scale pattern with 100 nm structure could be transcribed on the usual type of cover glass with a dyed resist. This fabrication technique can provide a disposable scale tool at a low cost, which is very suitable for calibrating the optical performance given by novel microscopy as well as the conventional one.