Monitoring of humoral immunity in 3–4 year-old anti-pertussis vaccinated children is one of the approaches for pertussis epidemiological surveillance. It’s a predictive indicator for comprehensive assessment of morbidity in the region. The agglutination reaction (RA) used earlier has been displaced with enzyme immunoassay (ELISA). So, two questions arose: regarding i) a relevance of using ELISA in whole-cell vaccinated individuals, and ii) correctness of large-scale data interpretation without applying a unified IgG reference value. Study aims were to assess humoral anti-pertussis population immunity by ELISA, to determine features of humoral protection in vaccinated subjects after using various vaccine types, to identify age features, to make recommendations for evaluating seromonitoring data. Blood serum samples from anti-pertussis vaccinated children in Samara region (n = 1729) were examined in 2016–2020; children were divided into 4 age groups ( 3, 3–4, 5–7, ≥ 7.1 years) and into 3 type vaccines groups: cellular, acellular and combined. We used two ELISA kits, which are designed to assess post-vaccination immunity, according to the manufacturer’s instructions, quantifying IgG againstB. pertussis, which differ in units and reference values. Statistical analysis was performed using StatTech program v.2.6.2. The largest and smallest percentage of seronegative children was in the cellular-vaccinated and acellular-vaccinated group, respectively. Seronegative percentage in the “indicator group” reaches 55.2% being significantly higher than acceptable 10.0%-level, and differs from the literature data (30–40%), which were obtained mainly with RA. This may suggest about an incorrect interpretation of post-vaccination immunity data evaluation strictly according to manufacturers’ reference criteria, especially for whole-cell vaccinated subjects. A method for calculating the seronegative percentage is proposed that relies on the prognostic negative data level as a correction factor. The required values were obtained during comparison studies of random serum samples from 3–4-year-old children (n = 70) using two ELISA kits (“RIDASCREEN®Bordetella”, “SeroPertussis™ IgG”) and intra-laboratory comparison panel. It should be given preference for sets with a greater prognostic negative data level, if population seronegative percentage is assessed.