Sensitive and quantitative detection of protein biomarkers with a point-of-care (POC) assay is significant for early diagnosis, treatment, and prognosis of diseases. In this paper, a quantitative lateral flow assay with high sensitivity for protein biomarkers was established by utilizing fluorescent nanospheres (FNs) as reporters. Each fluorescent nanosphere (FN) contains 332 ± 8 CdSe/ZnS quantum dots (QDs), leading to its superstrong luminescence, 380-fold higher than that of one QD. Then a detection limit of 27.8 pM C-reaction protein (CRP) could be achieved with an immunofluorescent nanosphere (IFN)-based lateral flow test strip. The assay was 257-fold more sensitive than that with a conventional Au-based lateral flow test strip for CRP detection. Besides, the fluorescence intensity of FNs and bioactivity of IFNs were stable during 6 months of storage. Hence, the assay owns good reproducibility (intra-assay variability of 5.3% and interassay variability of 6.6%). Furthermore, other cancer biomarkers (PSA, CEA, AFP) showed negative results by this method, validating the excellent specificity of the method. Then the assay was successfully applied to quantitatively detect CRP in peripheral blood plasma samples from lung cancer and breast cancer patients, and healthy people, facilitating the diagnosis of lung cancer. It holds a good prospect of POC protein biomarker detection.