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Starch is a polysaccharide that is abundantly found in nature and is generally used as an energy source and energy storage in many biological and environmental processes. Naturally, starch tends to be in miniscule amounts, creating a necessity for quantitative analysis of starch in low-concentration samples. Existing studies that are based on the spectrophotometric detection of starch using the colorful amylose–iodine complex lack a detailed description of the analytical procedure and important parameters. In the present study, this spectrophotometry method was optimized, tested, and applied to studying starch content of atmospheric bioaerosols such as pollen, fungi, bacteria, and algae, whose chemical composition is not well known. Different experimental parameters, including pH, iodine solution concentrations, and starch solution stability, were tested, and method detection limit (MDL) and limit of quantification (LOQ) were determined at 590 nm. It was found that the highest spectrophotometry signal for the same starch concentration occurs at pH 6.0, with an iodine reagent concentration of 0.2%. The MDL was determined to be 0.22 μg/mL, with an LOQ of 0.79 μg/mL. This optimized method was successfully tested on bioaerosols and can be used to determine starch content in low-concentration samples. Starch content in bioaerosols ranged from 0.45 ± 0.05 (in bacteria) to 4.3 ± 0.06 μg/mg (in fungi).