Background Screening using dipstick urinalysis has long been performed in 3‐year‐old children; however, it is ineffective in detecting congenital anomalies of the kidney and urinary tract (CAKUT). Measurement of the urinary β2‐microglobulin (β2MG)/creatinine (Cr) ratio may be more effective for this purpose. Analysis of dried urine spots (DUS) on filter paper is suitable for mass screening since operational costs are low and samples are easy to collect and transport. We examined the accuracy of measuring the urinary β2MG/Cr ratio in DUS on filter paper. Methods We collected 2,623 urine samples from 3‐year‐old children. β2MG and Cr levels were measured in DUS on filter paper. We examined the correlation between the β2MG/Cr ratios measured in DUS and using the conventional method in 640 samples using the coefficient of determination test. Children with high β2MG/Cr ratios (>0.6 µg/mg Cr) in DUS samples were further examined to establish a definitive diagnosis. Results There was strong correlation between the two methods for determination of β2MG levels (r2 = 0.68; P < 0.001) and β2MG/Cr ratios (r2 = 0.69; P < 0.001). Of the 2,623 children, 38 (1.45%) had β2MG/Cr ratios >0.6. Thirty‐five children were subsequently examined, resulting in findings of renal hypodysplasia (n = 2, 0.08%), horseshoe kidney (n = 1, 0.04%), renal tubular dysfunction with hepatoblastoma (n = 1, 0.04%), data abnormality (high urine β2MG level, n = 6, 0.23%; high serum Cr level, n = 1, 0.04%), and normal values (n = 24, 0.91%). Conclusions We evaluated a practical method for measuring β2MG/Cr ratios in DUS as a screening method to detect CAKUT in 3‐year‐old children.