Objective To improve and establish a molecular fluorescencespectrophotometry method for determining the content of vitamin ${\mathrm{B}}_{2}$ in animal derived foods.Methods Using pork belly, chicken, pig liver, and cow kidney as researchobjects, and referring to GB 5009.85-2016 National Standard of FoodSafety-Determination of Vitamin ${B}_{2}$ in Foods, further simplified theexperimental process, optimize experimental conditions, analyzed variousfactors that affect the results during the detection process, such as $\mathrm{{pH}}$ , light intensity, andthe effect of sodium hydrosulfite on fluorescence quenching, improvedthe accuracy of the method for determining vitamin ${\mathrm{B}}_{2}$ in animal derived foods.Results The maximum fluorescence intensity was determined at pH 6.5. Asthe light intensity increased and the light duration increased, thefluorescence intensity gradually decreased. After 5 hours, vitamin ${\mathrm{B}}_{2}$ basically decomposedcompletely. Vitamin ${\mathrm{B}}_{2}$ exhibited good linearitywithin the range of $0 \sim {10\mu}\mathrm{g}$ , with ${r}^{2}={0.9993}$ ; the limit of detection was ${0.004}\mathrm{{mg}}/{100}\mathrm{\;g}$ ,and the limit of quantification was ${0.02}\mathrm{{mg}}/{100}\mathrm{\;g}$ .The recovery rates for spiking ranged from ${90}\%\sim {100}\%$ . The relativestandard deviation of parallel sample determination results was lessthan 6%. Conclusion This method is accurate, simple in equipment, andcost-effective, and can meet the needs of grassroots laboratories forthe determination of vitamin ${\mathrm{B}}_{2}$ in animal derivedfoods.