Detection of allergen-induced basophil activation by flow cytometry has been shown to be a useful tool for allergy diagnosis. The aim of this study was to assess the potential of this technique for the diagnosis of pediatric house dust mite allergy. Quantification of total and specific IgE and basophil activation test were performed to evaluate mite allergic (n = 24), atopic (n = 23), and non-allergic children (n = 9). Allergen-induced basophil activation was detected as a CD63-upregulation. Receiver operating characteristics (ROC) curve analysis was performed to calculate the optimal cut-off value of activated basophils discriminating mite allergic and non-allergic children. ROC curve analysis yielded a threshold value of 18% activated basophils when mite-sensitized and atopic children were studied [area under the curve (AUC) = 0.99, 95% confidence interval (CI) = 0.97–1.01, p < 0.001] with a sensitivity and specificity of 96% for 16 μg/ml mite extract. Analysis of the data obtained with 1.6 μg/ml mite extract defined a cut-off value of 8% activated basophils (AUC = 0.96, 95% CI = 0.91–1.01; p < 0.001) with a sensitivity of 82% and specificity of 100%. Comparison between mite allergic and non-allergic children produced a cut-off of 8% activated basophils (AUC = 1.0) with 16 μg/ml allergen extract and a sensitivity and specificity of 100%. The same threshold and specificity values were obtained with 1.6 μg/ml extract (AUC = 97%, 95% CI = 0.92–1.02; p < 0.001) but sensitivity decreased to 83%. Two atopic children showed negative skin prick and basophil activation tests and high specific IgE (>43 kU/l) values for Dermatophagoides pteronyssinus allergen. They also showed positive prick (wheal diameter >1.0 cm) and basophil activation (>87%) tests and high specific IgE (>100 kU/l) with shrimp allergen. Shrimp sensitization was demonstrated by high levels of Pen a 1-specific IgE (>100 kU/l). Cross-reactivity between mite and shrimp was confirmed by fluorescence enzyme immunoassay (FEIA-CAP) inhibition study in these two cases. This study demonstrated that the analysis of allergen-induced CD63 upregulation by flow cytometry is a reliable tool for diagnosis of mite allergy in pediatric patients, with sensitivity similar to routine diagnostic tests and a higher specificity. Furthermore, this method can provide additional information in case of disagreement between in vivo and in vitro test results.