In this work, kiwi juice functioned as the reducing and capping reagent to obtain stable silver nanoparticles (AgNPs), which was very effective and inexpensive. Importantly, it could be finished within 2 min at room temperature, making the synthesis much faster and easier. Compared with the most common AgNPs (such as sodium citrate coated AgNPs), the proposed AgNPs presented excellent properties such as high stability. Moreover, a new analytical assay was established for protease K with the colour turning from light yellow to brown when protease K was introduced into the AgNPs aqueous solution. It was found that the increase of AgNPs absorbance at 470 nm was proportional to the concentration of protease K in the range of 0.50–15 μg mL⁻¹ with the limit of detection as low as 0.47 μg mL⁻¹. This selective and colorimetric assay has a great application prospect in bioanalysis due to its simplicity and specificity for protease K detection.