A novel security-enhanced scheme combining improved deoxyribonucleic acid (DNA) encoding encryption at the bit-level with matrix scrambling at the symbol-level is proposed in OFDM-PON for the first time in this paper. In our proposed scheme, firstly each subcarrier is encrypted by improved DNA encoding encryption, which includes the functioning of key base series and the cross interchange. And the selected encoding rules, decoding rules, key base series, operating principles and the positions of cross interchange are dynamically changing, which enhances the robustness against malicious attacks by illegal attackers. Then during the matrix scrambling process, the non-equal-length quadrature amplitude modulation (QAM) matrix is divided into several squares of equal length according to an optimum method. At the same time, the times of matrix scrambling can be determined randomly. With the multi-fold encryption of the proposed scheme, the achieved key space can reach up to 10154, which can sufficiently ensure the physical layer security. Experimental verification of the proposed security-enhanced strategy was demonstrated in an 8 Gb/s 16QAM orthogonal frequency division multiplexing passive optical network (OFDM-PON) system over 25-km standard single-mode fiber (SSMF). The experimental results prove that the two-level coordinated encryption at the bit-level and symbol-level using chaos and encryption can effectively protect data from violent attacks, differential attacks, etc.