Background: As the standard first-line treatment for NSCLC patients with EGFR-sensitive mutation, epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) were proved to be highly effective and less toxic compared to traditional chemotherapeutic regimens. But little is known about the correlation between tumor-infiltrating immune cells in the tumor microenvironment and clinical outcomes. This study was designed to investigate the association between tumor-infiltrating immune cells and the clinical outcome after first-line targeted therapy in EGFR exon 21 mutated lung adenocarcinoma (LUAD) patients. Methods: A total of 26 advanced LUAD patients with EGFR exon 21 mutation who received first-generation EGFR-TKIs as initial therapy between January 2017 and October 2019 were included in this study. All the patients were treated at Affiliated Dongguan Hospital, Southern Medical University, China. Patients who progressed within 12 months after first-line EGFR-TKIs therapy were defined as experiencing early progression (EP), and patients who progressed after 12 months or without progression were defined as experiencing late progression (LP). We analyzed immune markers in biopsy from these patients before treatment with EGFR-TKIs and examined the infiltration of immune cells and expression of PD-L1 in immune cells using fluorescent multiplex immunohistochemistry (mIHC) stained with CD8/CD56/CD68/CD163/FOXP3/PD-L1 antibodies. Results: In this cohort, we observed that after first-line EGFR-TKIs therapy in EGFR exon 21 mutated LUAD patients, the density of CD8+ FOXP3+T cells infiltrated in tumor area of LP group was significantly lower than EP group (P = 0.038). However, the difference of the density of the other infiltrated immune cells including CD8+T cells, FOXP3+Treg cells, CD8+PD-L1+T cells, CD68+CD163+M2macrophages, etc., were not significant between LP group and EP group (P > 0.05). The “optimal” cutoff value for CD8+ FOXP3+T cells infiltrated in tumor area was 5.45 (n/mm2) using the survminer package in R software according to PFS. Kaplan-Meier analysis suggested that the density of CD8+ FOXP3+T tell infiltrated in tumor area contributed to risk factor for PFS (HR = 5.56, P = 0.00054). Taking 5.45 (n/mm2) as the cutoff value, the percent of LP patients in low density of CD8+ FOXP3+T cells infiltrated in tumor area group was close to being statistically significant higher than high density of CD8+ FOXP3+T cells group (47% vs. 0%. P = 0.058). Conclusions: This study indicated that the low density of CD8+FOXP3+ T cells infiltrated in tumor area was associated with favorable clinical outcome in EGFR exon 21 mutated LUAD patients treated with first-generation EGFR-TKIs at first-line.