The invention discloses a sampling method for non-damage fixed position observation of plant stomata in a field experiment, which comprises the following steps of: according to experimental design, selecting functional leaves, avoiding overripe leaves, over tender leaves, leaves damaged by diseases and insects and malformed leaves, and cleaning object leaves; evenly coating a thin layer of nail polish at a position to be observed, and natural drying or blowing to be nearly dry by using a washing ear ball; flatly and tightly pasting a transparent adhesive tape on a clean glass slide, using a dissecting needle or a clean blade for slowly guiding to prevent air bubbles, and tightly pasting the adhesive tape with the width being at least 2mm around the nail polish on the glass slide; and numbering, clean packaging, and taking back to the laboratory for microscopic observation and measurement. The method has the advantages of being capable of bulk sampling in field living leaves, having no damage to the leaves, fixed position recording stomatal conditions, observing daily change of the stomata of the leaves under different processing conditions, and the like, and can be used for the research and practice in the fields such as bioecology, plant physiology, plant germplasm resource, plant thremmatology, cultivation science, etc.