The invention relates to a preparation method for a nicotine standard substance. The preparation method for the nicotine standard substance comprises the following steps: a, preparing coarse nicotine: adding ground waste tobacco into sodium hydroxide aqueous solution, stirring in water bath and extracting; filtering and adjusting the pH value of the filtrate by using sodium hydroxide to obtain nicotine extracting solution; distilling and concentrating the nicotine extracting solution, taking out the concentrated solution, cooling, adding sodium hydroxide aqueous solution and adjusting the pH value to be 11 to obtain the concentrated distillate; adding the concentrated distillate into chloroform, extracting and dehydrating the extracting solution by using anhydrous sodium sulfate to obtain the coarse nicotine solution; and b, preparing pure nicotine: performing reduced-pressure distillation and purification on the coarse nicotine obtained in the step a or the commercial coarse nicotine with the purity of not less than 60 percent under the conditions of pressure of 4.5-6.5 kPa and temperature of 160-170 DEG C to obtain pure nicotine; and subpackaging into a plurality of brown ampoule bottles of 1.0 mL under the protection of nitrogen and storing in a refrigerator at the temperature of 0 to 4 DEG C in dark place.